U.S. patent application number 15/841707 was filed with the patent office on 2018-05-10 for treatment for exposure to nerve agent.
The applicant listed for this patent is Georgetown University. Invention is credited to Esther H. CHANG, Kathleen F. PIROLLO.
Application Number | 20180126004 15/841707 |
Document ID | / |
Family ID | 51621094 |
Filed Date | 2018-05-10 |
United States Patent
Application |
20180126004 |
Kind Code |
A1 |
CHANG; Esther H. ; et
al. |
May 10, 2018 |
TREATMENT FOR EXPOSURE TO NERVE AGENT
Abstract
The present application provides methods of preventing and
treating the toxic effects of exposure to organophosphate agents.
In embodiments, targeted cationic liposome complexes delivering
nucleic acid molecules encoding butyrylcholinesterase and a
polyproline rich peptide are administered. Suitably, the
administration is via inhalation or via aerosol. Also provided are
cationic liposome complexes and methods of making the complexes for
such administration.
Inventors: |
CHANG; Esther H.; (Potomac,
MD) ; PIROLLO; Kathleen F.; (Rockville, MD) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Georgetown University |
Washington |
DC |
US |
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|
Family ID: |
51621094 |
Appl. No.: |
15/841707 |
Filed: |
December 14, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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14208187 |
Mar 13, 2014 |
9878055 |
|
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15841707 |
|
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61783001 |
Mar 14, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/18 20130101; A61K
9/1272 20130101; A61K 48/005 20130101; A61K 47/6913 20170801 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 9/18 20060101 C12N009/18; A61K 9/127 20060101
A61K009/127 |
Claims
1. A method of delivering butyrylcholinesterase (BChE) across the
blood-brain barrier and also to the bloodstream of a mammal,
comprising administering to the mammal a cationic liposome complex,
wherein the cationic liposome complex comprises: (a) a cationic
liposome; (b) an anti-transferrin receptor single chain Fv
(TfRscFv) directly complexed with, but not chemically conjugated
to, the cationic liposome; (c) a nucleic acid molecule encoding
butyrylcholinesterase (BChE); and (d) a nucleic acid molecule
encoding a polyproline rich peptide, wherein the TfRscFv and the
cationic liposome are present at a ratio in the range of about 1:20
to about 1:40 (w:w) and the nucleic molecules are present at a
ratio of about 1:5 to about 1:20 (.mu.g nucleic acid:.mu.g
liposome).
2. The method of claim 1, wherein the cationic liposome complex is
administered via a route selected from the group consisting of
intranasal administration, intravenous administration, oral
administration, sublingual administration, intramuscular
administration, intralesional administration, intradermal
administration, transdermal administration, intraocular
administration, intraperitoneal administration, percutaneous
administration, aerosol administration, intraorgan administration,
intracereberal administration, topical administration, subcutaneous
administration, endoscopic administration, slow release implant,
administration via an osmotic or mechanical pump and administration
via inhalation.
3. The method of claim 1, wherein the cationic liposome complex is
administered via transdermal administration, intranasal
administration or via inhalation.
4. The method of claim 1, wherein the cationic liposome complex is
administered so as to result in an amount of BChE in the
bloodstream of a human of at least 250 mg/70 kg (weight of
BChE/weight of the human).
5. The method of claim 1, wherein the nucleic acid molecule
encoding butyrylcholinesterase (BChE) is contained in a first
plasmid associated with the cationic liposome; and the nucleic acid
molecule encoding the polyproline rich peptide is contained in a
second plasmid associated with the cationic liposome.
6. The method of claim 1, wherein the nucleic acid molecule
encoding butyrylcholinesterase (BChE) and the nucleic acid molecule
encoding the polyproline rich peptide are contained in the same
plasmid associated with the cationic liposome.
7. The method of claim 5, wherein the nucleic acid molecule
encoding BChE is contained in the first plasmid construct,
comprising, from 5' to 3': (a) at least one human adenovirus
enhancer sequence; (b) a cytomegalovirus (CMV) promoter; (c) a
multiple cloning site; (d) the nucleic acid molecule encoding BChE;
and (e) an SV 40 poly A sequence, wherein the 3' end of the plasmid
construct does not comprise adenovirus map units 9-16 when compared
to a wild-type adenovirus.
8. The method of claim 5, wherein the nucleic acid molecule
encoding the polyproline rich peptide is contained in the second
plasmid construct, comprising, from 5' to 3': (a) at least one
human adenovirus enhancer sequence; (b) a cytomegalovirus (CMV)
promoter; (c) a multiple cloning site; (d) the nucleic acid
molecule encoding the polyproline rich peptide; and (e) an SV 40
poly A sequence, wherein the 3' end of the plasmid construct does
not comprise adenovirus map units 9-16 when compared to a wild-type
adenovirus.
9. The method of claim 6, wherein the nucleic acid molecule
encoding butyrylcholinesterase (BChE) and/or the nucleic acid
molecule encoding the polyproline rich peptide are contained in a
plasmid, comprising one or more inserts, each insert comprising
from 5' to 3': (a) at least one human adenovirus enhancer sequence;
(b) a cytomegalovirus (CMV) promoter; (c) a multiple cloning site;
(d) one or more nucleic acid molecules encoding the nucleic acid
molecule encoding butyrylcholinesterase (BChE) and/or one or more
nucleic acid molecules encoding the polyproline rich peptide; and
(e) an SV 40 poly A sequence, wherein the 3' end of the plasmid
construct does not comprise adenovirus map units 9-16 when compared
to a wild-type adenovirus.
10. The method of claim 1, wherein the BChE is a mutant version of
BChE.
11. The method of claim 10, wherein the mutant version of BChE is a
G117H mutant.
12. The method of claim 1, wherein the cationic liposome comprises
a mixture of one or more cationic lipids and one or more neutral or
helper lipids
13. The method of claim 12, wherein said cationic liposome
comprises a mixture of dioleoyltrimethylammonium phosphate with
dioleoylphosphatidylethanolamine and cholesterol; a mixture of
dioleoyltrimethylammonium phosphate with cholesterol, a mixture of
dimethyldioctadecylammonium bromide with
dioleoylphosphatidylethanolamine and cholesterol, a mixture of
dimethyldioctadecylammonium bromide with
dioleoylphosphatidylethanolamine, a mixture of
dimethyldioctadecylammonium bromide with cholesterol, or a mixture
of dioleoyltrimethylammonium phosphate with
dioleoylphosphatidylethanolamine.
14. The method of claim 1, wherein the nucleic acid molecules are
present at a molar ratio of about 10:1 to about 1:10 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding the polyproline rich peptide).
15. The method of claim 14, wherein the nucleic acid molecules are
present at a molar ratio of about 5:1 to about 1:5 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding the polyproline rich peptide).
16. The method of claim 14, wherein the nucleic acid molecules are
present at a molar ratio of about 4:1 (moles nucleic acid molecule
encoding butyrylcholinesterase (BChE):moles nucleic acid molecule
encoding the polyproline rich peptide), or wherein the nucleic acid
molecules are present at a molar ratio of about 2:1 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding the polyproline rich peptide).
17. The method of claim 14, wherein the nucleic acid molecules are
present at a molar ratio of about 1:1 (moles nucleic acid molecule
encoding butyrylcholinesterase (BChE):moles nucleic acid molecule
encoding the polyproline rich peptide).
18. The method of claim 1, wherein the nucleic acid molecules are
encapsulated within the cationic liposome, contained within a
hydrocarbon chain region of the cationic liposome, associated with
an inner or outer monolayer, or any combination thereof.
19. A method of delivering butyrylcholinesterase (BChE) across the
blood-brain barrier and to the bloodstream of a human, comprising
administering intranasally or via aerosol inhalation to the mammal
a cationic liposome complex, wherein the cationic liposome complex
comprises: (a) a cationic liposome; (b) an anti-transferrin
receptor single chain Fv (TfRscFv) directly complexed with, but not
chemically conjugated to, the cationic liposome; (c) a nucleic acid
molecule encoding butyrylcholinesterase (BChE) associated with the
cationic liposome; and (d) a nucleic acid molecule encoding a
polyproline rich peptide associated with the cationic liposome,
wherein the TfRscFv and the cationic liposome are present at a
ratio in the range of about 1:20 to about 1:40 (w:w) and the
nucleic molecules are present at a ratio of about 1:5 to about 1:20
(.mu.g nucleic acid:.mu.g liposome), wherein the nucleic acid
molecules are present at a molar ratio of about 10:1 to about 1:10
(moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide), and wherein the complex is administered so as to result
in an amount of BChE in the bloodstream of the human of at least
250 mg/70 kg (weight of BChE/weight of the human).
20. The method of claim 19, wherein the BChE is a mutant version of
BChE; and wherein the cationic liposome comprises a mixture of
dioleoyltrimethylammonium phosphate with
dioleoylphosphatidylethanolamine and cholesterol; a mixture of
dioleoyltrimethylammonium phosphate with cholesterol, a mixture of
dimethyldioctadecylammonium bromide with
dioleoylphosphatidylethanolamine and cholesterol, a mixture of
dimethyldioctadecylammonium bromide with
dioleoylphosphatidylethanolamine, a mixture of
dimethyldioctadecylammonium bromide with cholesterol, or a mixture
of dioleoyltrimethylammonium phosphate with
dioleoylphosphatidylethanolamine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. 14/208,187, filed Mar. 13, 2014 (pending),
which claims benefit of U.S. Provisional Application No.
61/783,001, filed Mar. 14, 2013, the disclosures of each of which
are incorporated by reference herein in their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 12, 2017, is named 2474-0021US2_SL.txt and is 1,093 bytes
in size.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present application provides methods of preventing and
treating the toxic effects of exposure to organophosphate agents.
In embodiments, targeted cationic liposome complexes delivering
nucleic acid molecules encoding butyrylcholinesterase and nucleic
acid molecules encoding a polyproline rich sequence are
administered. Suitably, the administration is via inhalation. Also
provided are cationic liposome complexes and methods of making the
complexes for such administration.
Background of the Invention
[0004] Organophosphate agents (OP) are commonly used as pesticides,
insecticides and drugs for treatment of medical conditions such as
glaucoma and Alzheimer's disease. Unfortunately, they have also
been developed for use as nerve agents such as sarin, soman, VX,
and tabun. These OP compounds are among the most toxic chemicals
known. Exposure to even small amounts can be fatal. Death occurs
from asphyxiation resulting from paralysis of the diaphragm and
intracostal muscles, depression of the brain respiratory center,
bronchospasm, convulsions and excessive salivation (reviewed in 1).
The mechanism of OP poisoning is the phosphorylation of the serine
hydroxyl group in the active site of Acetylcholinesterase (AchE)
which leads to irreversible inactivation of the enzyme. AchE, which
hydrolyzes Acetylcholine (Ach) at the synaptic space, is an
essential enzyme in neurotransmission. Inactivation of AchE results
in a rapid buildup of Ach subsequently producing PNS cholinergic
hyperstimulation and death. In the CNS, cholinergic
hyperexcitability increases neuronal firing triggering convulsions
and acute neuronal cell death.
[0005] Although there are antidotal treatments for post-exposure
use, they have shown limited efficacy, produce serious side
effects, and do not prevent incapacitation (transient or permanent)
or irreversible brain damage (1,2). Thus, prophylactic measures are
being sought. One approach for counteracting OP toxicity is the use
of a bioscavenger to sequester and neutralize these compounds. Of
the bioscavengers tested, human serum butyrylcholinesterase (BChE)
(also called pseudocholinesterase, or cholinesterase) appears to be
the most suited for human use (3). BChE is a serine enzyme present
in almost every tissue including plasma, brain, muscle, kidney,
liver and lung (4). Human BChE (hBChE) (340 kDa) in serum is a
globular tretrameric molecule with a T.sub.1/2 of 11-14 days and is
composed of four identical subunits and is protected from
proteolysis through heavy glycosylation (5). The assembly of the
individual subunits into the tetramer requires the presence of a
polyproline rich peptide derived from lamellipodin (5) or from rat
collagen tail (AChE Q subunit) (Bon paper, Krejci paper, Antamirano
paper), or any other polyproline rich protein. BChE is naturally
expressed at relatively high levels, 4 times that of the average
gene (4). It also plays an important role in the degradation of
numerous ester-containing drugs and is a natural bioscavenger of
cholinesterase inhibitors, including potent OP nerve agents.
[0006] Each molecule of hBChE neutralizes one molecule of OP (6).
It has been reported that pretreatment with recombinant human BChE
and human serum BChE could protect animals (including rodents,
guinea pigs, pigs and non-human primates) from up to 5 times
LD.sub.50 of nerve agents (6,7). The irreversible binding and
inactivating function of BChE with a broad spectrum of OP poisons
make it an ideal candidate for a prophylactic treatment against
nerve agents. In addition to its use for a variety of wartime pre-
and post-exposure scenarios, it also has potential use as a
pretreatment for first responders reacting to
intentional/accidental nerve gas release and as a post-exposure
therapy for pesticide overexposure, cocaine overdose, or
succinyl-choline-induced apnea (8).
[0007] It has been estimated that in a human, a BChE dose of 250
mg/70 kg is required to achieve efficient protection following a
challenge with one LD.sub.50 of OPs (3). However, the naturally
occurring amount of this bioscavenger enzyme in blood (.about.8-72
mg/6 L) is too low to achieve adequate protection due to the
stoichometric and irreversible binding of, and the interaction
between, the OP and BChE; the unfavorable OP/BChE mass ratio; and
the aging of the enzyme (4,9). Thus, it is critical to develop a
means to significantly increase the level of BChE expression and
amount in plasma. Toward this end, different strategies are being
developed. The most straightforward is the direct injection of a
large dose of highly purified natural hBChE to increase the amount
in the bloodstream. This has proven to be successful for protection
against lethal doses of soman and VX but, is not practical for
battlefield use. Moreover, use of transgenic animals and cell
culture has not been able to produce sufficient quantities of hBChE
to be practical and feasible for use.
[0008] Derivation of BChE mutants capable of reactivating
spontaneously (making them available to bind and deactivate
additional molecules of OP) is another approach being employed with
some success. BChE was shown to gain OP hydrolase activity and
increased reactivation when a Histidine was substituted for Glycine
at position 117 (G117H) (10,11). This mutant is efficient at
hydrolyzing the acetylcholinesterase inhibitor echothiophate and
can also efficiently hydrolyze the nerve agents sarin and VX (9).
More importantly, transgenic mice expressing the G117H mutant are
resistant to OP (12). Although upwards of 60 BChE mutants have been
produced, the G117H remains one of the most efficient and studied
to date (9). However, as yet, no means of efficiently delivering or
producing for extended periods of time in the body after
administration, a BChE or tetrameric form of active mtBChE via
non-invasive routes, has been developed.
[0009] There is, therefore, an urgent need to develop technologies
and methods to deliver BChE for prevention and treatment of
exposure to OP agents. The present invention fulfills these needs
by providing cationic-liposome-based drug delivery systems for such
treatment and/or prevention.
BRIEF SUMMARY OF THE INVENTION
[0010] In one embodiment, methods of treating or preventing
toxicity associated with exposure to an organophosphate agent in a
mammal are provided. Such methods suitably comprise administering
to the mammal a cationic liposome complex, wherein the cationic
liposome complex comprises a cationic liposome, a ligand directly
complexed with, but not chemically conjugated to, the cationic
liposome, a nucleic acid molecule encoding butyrylcholinesterase
(BChE) associated with the cationic liposome, and a nucleic acid
molecule encoding a polyproline rich peptide associated with the
cationic liposome.
[0011] In embodiments, the complex is administered via a route
selected from the group consisting of intranasal administration,
intravenous administration, oral administration, sublingual
administration, intramuscular administration, intralesional
administration, intradermal administration, transdermal
administration, intraocular administration, intraperitoneal
administration, percutaneous administration, aerosol
administration, intraorgan administration, intracereberal
administration, topical administration, subcutaneous
administration, endoscopic administration, slow release implant,
administration via an osmotic or mechanical pump and administration
via inhalation.
[0012] Suitably, the ligand is transferrin, an antibody and an
antibody fragment, including a single chain Fv antibody fragment,
such as an anti-transferrin receptor single chain Fv (TfRscFv).
[0013] In further embodiments, the ligand-targeted cationic
liposome further comprises a peptide comprising a
K[K(H)KKK]5-K(H)KKC (HoKC) (SEQ ID NO: 1) peptide associated with
the cationic liposome.
[0014] Suitably, the nucleic acid molecule encoding BChE is
contained in a first plasmid and the nucleic acid molecule encoding
the polyproline rich peptide is contained in a second plasmid. In
embodiments, the nucleic acid molecule encoding BChE is contained
in a first plasmid construct, comprising, from 5' to 3': (a) at
least one human adenovirus enhancer sequence; (b) a cytomegalovirus
(CMV) promoter; (c) a multiple cloning site; (d) the nucleic acid
molecule encoding BChE; and (e) an SV 40 poly A sequence, wherein
the 3' end of the plasmid construct does not comprise adenovirus
map units 9-16 when compared to a wild-type adenovirus. In
embodiments, the nucleic acid molecule encoding the polyproline
rich peptide is contained in a second plasmid construct,
comprising, from 5' to 3': (a) at least one human adenovirus
enhancer sequence; (b) a cytomegalovirus (CMV) promoter; (c) a
multiple cloning site; (d) the nucleic acid molecule encoding the
polyproline rich peptide; and (e) an SV 40 poly A sequence, wherein
the 3' end of the plasmid construct does not comprise adenovirus
map units 9-16 when compared to a wild-type adenovirus.
[0015] Alternatively the nucleic acid molecule encoding BChE, and
the nucleic acid molecule encoding the polyproline rich peptide,
are in the same construct, but the nucleic acid molecule encoding
BChE is placed downstream from the high expression promoter
disclosed in U.S. Published Patent Application No. 2007/0065432,
while the nucleic acid molecule encoding the polyproline rich
peptide is placed downstream of a standard promoter such as RSV or
CMV
[0016] Suitably, the BChE is a mutant version of BChE, including
the mutant version of BChE is a G117H mutant.
[0017] In exemplary embodiments, the cationic liposome comprises a
mixture of one or more cationic lipids and one or more neutral or
helper lipids. Suitably the ligand and the cationic liposome are
present at a ratio in the range of about 1:1 to about 1:100 (w:w),
for example at a ratio in the range of about 1:10 to about 1:50
(w:w), or at a ratio in the range of about 1:20 to about 1:40
(w:w).
[0018] In embodiments, the cationic liposome comprises a mixture of
dioleoyltrimethylammonium phosphate with
dioleoylphosphatidylethanolamine and cholesterol; a mixture of
dioleoyltrimethylammonium phosphate with cholesterol, a mixture of
dimethyldioctadecylammonium bromide with
dioleoylphosphatidylethanolamine and cholesterol, a mixture of
dimethyldioctadecylammonium bromide with
dioleoylphosphatidylethanolamine, a mixture of
dimethyldioctadecylammonium bromide with cholesterol, or a mixture
of dioleoyltrimethylammonium phosphate with
dioleoylphosphatidylethanolamine.
[0019] Suitably, the nucleic acid molecules in the cationic
immunoliposome complex are present at a molar ratio of about 10:1
to about 1:10 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide), or the nucleic acid molecules are
present at a molar ratio of about 5:1 to about 1:5 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding a polyproline rich peptide), or more
suitably the nucleic acid molecules are present at a molar ratio of
about 4:1 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide), or more suitably the nucleic acid
molecules are present at a molar ratio of about 2:1 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding a polyproline rich peptide) or more suitably
the nucleic acid molecules are present at a molar ratio of about
1:1 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide).
[0020] In embodiments, the total amount of nucleic acid molecules
is present at a weight ratio of between about 1:1 to about 1:40
(.mu.g nucleic acid:.mu.g liposome), or about 1:5 to about 1:20
(.mu.g total nucleic acid:.mu.g liposome), or more suitably the
total amount of nucleic acid molecules is present at a weight ratio
about 1:10 (.mu.g total nucleic acid:.mu.g liposome).
[0021] Suitably, the complex is administered so as to treat
toxicity associated with exposure to at least 1.times.LD.sub.50 of
the organophosphate agent, more suitably so as to treat toxicity
associated with exposure to up to 10.times.LD.sub.50 of the
organophosphate agent. In embodiments, the complex is administered
so as to prevent toxicity associated with exposure to at least
1.times.LD.sub.50 of the organophosphate agent, more suitably
exposure to up to 10.times.LD50 of the organophosphate agent.
[0022] In embodiments, the complex is administered immediately
before or after exposure to the organophosphate agent. In further
embodiments, the complex is administered at least 6 hours prior to
potential exposure to the organophosphate agent. Suitably, the
complex is administered at least once a week prior to potential
exposure to the organophosphate agent.
[0023] Suitably, the mammal is a human.
[0024] Also provided are methods of treating toxicity associated
with exposure to an organophosphate agent in a human. Such methods
suitably comprise administering intranasally or via aerosol
inhalation to the human a cationic liposome complex, wherein the
cationic liposome complex comprises a cationic liposome, an
anti-transferrin receptor single chain Fv (TfRscFv) directly
complexed with, but not chemically conjugated to, the cationic
liposome, a nucleic acid molecule encoding butyrylcholinesterase
(BChE) contained in a first plasmid associated with the cationic
liposome, and a nucleic acid molecule encoding a polyproline rich
peptide contained in a second plasmid associated with the cationic
liposome. Suitably the TfRscFv and the cationic liposome are
present at a ratio in the range of about 1:20 to about 1:40 (w:w)
and the nucleic molecules are present at a ratio of about 1:5 to
about 1:20 (.mu.g nucleic acid:.mu.g liposome). Suitably, the
nucleic acid molecules in the cationic immunoliposome complex are
present at a molar ratio of about 10:1 to about 1:10 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE): moles nucleic
acid molecule encoding a polyproline rich peptide), or the nucleic
acid molecules are present at a molar ratio of about 5:1 to about
1:5 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide), or more suitably the nucleic acid molecules are present
at a molar ratio of about 4:1 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide). or more suitably the nucleic acid
molecules are present at a molar ratio of about 2:1 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding a polyproline rich peptide) or more suitably
the nucleic acid molecules are present at a molar ratio of about
1:1 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide). In embodiments, the complex is administered so as to
treat toxicity associated with exposure to at least 1.times.LD50 of
the organophosphate agent.
[0025] Also provided are methods of preventing toxicity associated
with exposure to an organophosphate agent in a human. Such methods
suitably comprise administering intranasally or via aerosol
inhalation to the human a cationic liposome complex, wherein the
cationic liposome complex comprises a cationic liposome, an
anti-transferrin receptor single chain Fv (TfRscFv) directly
complexed with, but not chemically conjugated to, the cationic
liposome, a nucleic acid molecule encoding butyrylcholinesterase
(BChE) contained in a first plasmid associated with the cationic
liposome, and a nucleic acid molecule encoding a polyproline rich
peptide contained in a second plasmid associated with the cationic
liposome. Suitably the TfRscFv and the cationic liposome are
present at a ratio in the range of about 1:20 to about 1:40 (w:w)
and the nucleic molecules are present at a ratio of about 1:5 to
about 1:20 (.mu.g nucleic acid:.mu.g liposome). Suitably, the
nucleic acid molecules in the cationic immunoliposome complex are
present at a molar ratio of about 10:1 to about 1:10 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE): moles nucleic
acid molecule encoding a polyproline rich peptide), or the nucleic
acid molecules are present at a molar ratio of about 5:1 to about
1:5 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide), or more suitably the nucleic acid molecules are present
at a molar ratio of about 4:1 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide) or more suitably the nucleic acid
molecules are present at a molar ratio of about 2:1 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding a polyproline rich peptide) or more suitably
the nucleic acid molecules are present at a molar ratio of about
1:1 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide). In embodiments, the complex is administered so as to
prevent toxicity associated with exposure to at least 1.times.LD50
of the organophosphate agent.
[0026] Also provided are methods of delivering
butyrylcholinesterase (BChE) to the bloodstream of a mammal. Such
methods suitably comprise administering intranasally or via aerosol
inhalation to the mammal a cationic liposome complex, wherein the
cationic liposome complex comprises a cationic liposome, an
anti-transferrin receptor single chain Fv (TfRscFv) directly
complexed with, but not chemically conjugated to, the cationic
liposome, a nucleic acid molecule encoding butyrylcholinesterase
(BChE) contained in a first plasmid associated with the cationic
liposome, and a nucleic acid molecule encoding a polyproline rich
peptide contained in a second plasmid associated with the cationic
liposome. Suitably the TfRscFv and the cationic liposome are
present at a ratio in the range of about 1:20 to about 1:40 (w:w)
and the nucleic molecules are present at a ratio of about 1:5 to
about 1:20 (.mu.g nucleic acid:.mu.g liposome). Suitably, the
nucleic acid molecules in the cationic immunoliposome complex are
present at a molar ratio of about 10:1 to about 1:10 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding a polyproline rich peptide), or the nucleic
acid molecules are present at a molar ratio of about 5:1 to about
1:5 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide), or more suitably the nucleic acid molecules are present
at a molar ratio of about 4:1 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide) or more suitably the nucleic acid
molecules are present at a molar ratio of about 2:1 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding a polyproline rich peptide) or more suitably
the nucleic acid molecules are present at a molar ratio of about
1:1 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE): moles nucleic acid molecule encoding a polyproline rich
peptide). In embodiments, the complex is administered so as to
result in an amount of BChE in the bloodstream of the human of at
least 250 mg/70 kg (weight of BChE/weight of the human).
[0027] Alternatively the nucleic acid molecule encoding BChE, and
the nucleic acid molecule encoding the polyproline rich peptide,
are in the same construct, but the nucleic acid molecule encoding
BChE is placed downstream from the high expression promoter
disclosed in U.S. Published Patent Application No. 2007/0065432
(incorporated by reference herein in its entirety), while the
nucleic acid molecule encoding the polyproline rich peptide is
placed downstream of a standard promoter such as RSV or CMV
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0028] FIG. 1 shows the presence of transgene expression in
metastatic tumors from targeted catonic immunoliposome
delivery.
[0029] FIG. 2 shows increase in protein expression as a result of
placing a gene under the control a high expression promoter as
described herein.
[0030] FIGS. 3A and 3B show scL delivery to the brain in Balb/C
mice injected with scL carrying either the pSCMV high expression
plasmid containing the GFP gene (FIG. 3A) or carrying a
fluorescently labeled oligonucleotide (FIG. 3B).
[0031] FIG. 4A-4B show brain sections of the liposome complex
targeting the TfR and carrying a plasmid encoding GFP (100 .mu.g
cDNA) injected via a catheterized jugular vein in rats.
[0032] FIG. 5 shows level of expression of the luciferase gene
either as Free (unencapsulated), or scL-encapsulated plasmid DNA,
after intranasal administration to Balb/c mice.
[0033] FIG. 6 shows Western analysis of total cellular protein
using an antibody that detects both ERKI and ERKII proteins.
[0034] FIG. 7 shows the results of the subcloning of wt-BChE,
mt-BChE and ppro genes into the pSCMV vector.
[0035] FIG. 8 shows the percent supercoiled of wt-BChE, mt-BChE and
ppro genes in pSCMV vectors.
[0036] FIG. 9 shows the level of expression of BChE in CHO-K1 cells
after transfection with increasing amounts of DNA in the
scL-mtBChE/ppro complex.
[0037] FIG. 10 shows BChE Expression 16 days after In Vitro
Transfection of A549 Cells with scL-mtBChE/ppro complex.
[0038] FIG. 11 shows BChE Expression 28 days after In Vitro
Transfection of A549 Cells with scL-mtBChE/ppro complex.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The terms "complex," "nanocomplex," "liposome complex" and
"nanoliposome" are used interchangeably throughout to refer to the
cationic liposomes described herein. Exemplary cationic liposomes
for use in the practice of the present invention and methods of
production thereof are disclosed in U.S. Published Patent
Application Nos. 2003/0044407 and 2007/0065499, the disclosures of
each of which are incorporated by reference herein in their
entireties.
[0040] As used herein the term "about" refers to the recited value,
as well as values within 10% of the recited value. For example,
"about 100 nm" includes the values of 90 nm to 110 nm, including
values in between this range.
[0041] As used herein the term "ligand" refers to any suitable
targeting moiety that can be either chemically conjugated to, or
directly associated/complexed with, but not chemically conjugated
to, the cationic liposomes. Exemplary ligands for use in the
practice of the present invention include, but are not limited to,
proteins (e.g., transferrin or folate), peptides (e.g., L-37 pA),
antibodies, antibody fragments (including Fab' fragments and single
chain Fv fragments) and sugars (e.g., galactose), as well as other
targeting molecules.
[0042] Exemplary methods and compositions in which the complexes in
accordance with embodiments of this invention are made by a simple
and efficient non-chemical conjugation are disclosed in U.S.
Published Patent Application Nos. 2003/0044407 and 2007/0065499.
Exemplary methods and compositions in which the complexes in
accordance with embodiments of this invention are made via chemical
conjugation are disclosed in U.S. Pat. No. 7,479,276. The
disclosures of each of these patents and patent applications are
incorporated by reference herein in their entireties.
[0043] In exemplary embodiments, a whole antibody or an antibody
fragment can be used as the ligand to make the complexes of this
invention. In a suitable embodiment, an antibody fragment is used,
including Fab fragments and single chain Fv fragments (scFv) of an
antibody. One suitable antibody is an anti-Transferrin receptor
(anti-TfR) monoclonal antibody, and a suitable antibody fragment is
an scFv based on an anti-TfR monoclonal antibody. A suitable
anti-TfR monoclonal antibody is 5E9 (see, e.g., Hayes, B. F., et
al., "Characterization of a Monoclonal Antibody (5E9) that Defines
a Human Cell Surface Antigen of Cell Activation," J. Immunol.
127:347-352 (1981); Batra, J. K., et al., "Single-chain
Immunotoxins Directed at the Human Transferrin Receptor Containing
Pseudomonas Exotoxin A or Diphtheria Toxin: Anti-TFR(Fv)-PE40 and
DT388-Anti-TFR(Fv)," Mol. Cell. Biol. 11:2200-2205 (1991) (the
disclosures of which are incorporated herein by reference). An scFv
based on the full anti-TfR monoclonal antibody contains the
complete antibody binding site for the epitope of the TfR
recognized by this MAb as a single polypeptide chain of approximate
molecular weight 26,000. An scFv is formed by connecting the
component VH and VL variable domains from the heavy and light
chains, respectively, with an appropriately designed peptide, which
bridges the C-terminus of the first variable region and N-terminus
of the second, ordered as either VH-peptide-VL or VL-peptide-VH.
Additional ligands, such as those described throughout, can also be
used in the practice of the present invention.
[0044] In one embodiment, a cysteine moiety is added to the
C-terminus of the scFv. Although not wishing to be bound by theory,
it is believed that the cysteine, which provides a free sulfhydryl
group, may enhance the formation of the complex between the
antibody and the liposome in both the chemically conjugated and
non-chemically conjugated embodiments. With or without the
cysteine, the protein can be expressed in E. coli inclusion bodies
and then refolded to produce the antibody fragment in active
form.
[0045] Unless it is desired to use a sterically stabilized liposome
in the formation of the complexes, a first step in making exemplary
non-chemically conjugated complexes of the present invention
comprise mixing a cationic liposome or combination of liposomes
with the antibody or antibody fragment of choice. A wide variety of
cationic liposomes are useful in the preparation of the complexes
of this invention. Published PCT application WO99/25320 (the
disclosure of which is incorporated by reference herein in its
entirety) describes the preparation of several cationic liposomes.
Examples of suitable liposomes include phosphatidylcholine (PC),
phosphatidylserine (PS) and those that comprise a mixture
dioleoyltrimethylammonium phosphate (DOTAP) and
dioleoylphosphatidylethanolamine (DOPE), a DOTAP and DOPE and/or
cholesterol (chol); a mixture of dimethyldioctadecylammonium
bromide (DDAB) and DOPE and/or chol, or a mixture of DDAB and DOPE.
The ratio of the lipids can be varied to optimize the efficiency of
uptake of the therapeutic molecule for the specific target cell
type. The liposome can comprise a mixture of one or more cationic
lipids and one or more neutral or helper lipids. A desirable ratio
of cationic lipid(s) to neutral or helper lipid(s) is about
1:(0.5-3), preferably 1:(1-2) (molar ratio).
[0046] Suitable ligands, for example, proteins/peptides, antibody
or antibody fragments, are those that will bind to the surface of
the target cell, and preferably to a receptor that is
differentially expressed on the target cell. The ligands are mixed
with the cationic liposome or polymer at room temperature and at a
ligand (e.g., protein):lipid ratio (weight:weight) in the range of
about 1:10 to about 1:50, suitably about 1:20 to about 1:40
(w:w).
[0047] The ligand (e.g., the protein/peptide, antibody or antibody
fragment) and the liposome are allowed to incubate at room
temperature for a short period of time, typically for about 10-15
minutes, then the mixture is mixed with a therapeutic or diagnostic
agent of choice. Examples of therapeutic molecules or agents which
can be complexed to the liposome complexes include genes, high
molecular weight DNA (genomic DNA), plasmid DNA, antisense
oligonucleotides, peptides, ribozymes, nucleic acids (including
siRNA, miRNA and antisense), small molecules, viral particles,
immunomodulating agents, contrast agents for imaging, proteins and
chemical agents.
[0048] The ligand (e.g., the protein/peptide, antibody or antibody
fragment) and liposome combination is mixed with the therapeutic
agent at a ratio in the range of about 0.5:1 to about 1:40 (.mu.g
of agent:nmol of total lipid), suitably about 1:10 to 1:20 (.mu.g
of agent:nmole of total lipid) and incubated at room temperature
for a short period of time, typically about 10 to 15 minutes. For
use in vivo, 50% dextrose or 50% sucrose is added to a final
concentration of 5-20% (V:V) and mixed by gentle inversion for 5-10
seconds, or for larger volumes rotated at 20-30 RPM for 1-2 minutes
The size of the liposome complex is typically within the range of
about 50-500 nm as measured by dynamic light scattering using a
Malvern ZETASIZER.RTM. 3000 or a Malvern ZETASIZER.RTM. NANO-ZS.
See U.S. Published Patent Application No. 2003/0044407 and U.S.
patent application Ser. No. 11/520,796, the disclosures of which
are incorporated by reference herein in their entireties.
[0049] In one embodiment of this invention, the liposome used to
form the complex is a sterically stabilized liposome. Sterically
stabilized liposomes are liposomes into which a hydrophilic
polymer, such as PEG, poly(2-ethylacrylic acid), or
poly(n-isopropylacrylamide (PNIPAM) has been integrated. Such
modified liposomes can be particularly useful when complexed with
therapeutic agents, as they typically are not cleared from the
bloodstream by the reticuloendothelial system as quickly as are
comparable liposomes that have not been so modified. To make a
sterically stabilized liposome complex of the present invention,
the order of mixing the antibody or antibody fragment, the liposome
and the therapeutic or diagnostic agent is reversed from the order
set forth above. In a first step, a cationic liposome as described
above is first mixed with a therapeutic agent as described above at
a ratio in the range of about 0.5:1 to about 1:40 (.mu.g of
agent:nmol of lipid), suitably about 1:10 to 1:20 (.mu.g of
agent:nmole of lipid). To this lipoplex is added a solution of a
PEG polymer in a physiologically acceptable buffer at a ratio of
about 0.1:100 (nmol of PEG:nmol of liposome), suitably, about
0.5:50, for example, about 1:40 (nmol of PEG:nmol of liposome). The
resultant solution is incubated at room temperature for a time
sufficient to allow the polymer to integrate into the liposome
complex. The ligand (e.g., protein/peptide, antibody or antibody
fragment) then is mixed with the stabilized liposome complex at
room temperature and at a ligand (e.g., protein):lipid ratio in the
range of about 1:5 to about 1:40 (w:w). For use in vivo, 50%
dextrose or 50% sucrose is added to a final concentration of 5-20%
(V:V) and mixed by gentle inversion for 5-10 seconds, or for larger
volumes rotated at 20-30 RPM for 1-2 minutes.
[0050] The liposomal complexes prepared in accordance with the
present invention can be formulated as a pharmacologically
acceptable formulation for in vivo administration. The complexes
can be combined with a pharmacologically compatible vehicle or
carrier. The compositions can be formulated, for example, for
intravenous administration to a mammal, for example a human patient
to be benefited by administration of the therapeutic molecule, or
other payload, in the complex. The complexes are of an appropriate
size so that they are distributed throughout the body following
i.v. administration. Alternatively, the complexes can be delivered
via other routes of administration, such as intratumoral (IT),
intralesional (IL), aerosal, percutaneous, oral, endoscopic,
topical, intramuscular (IM), intradermal (ID), intraocular (IO),
intraperitoneal (IP), transdermal (TD), intranasal (IN),
intracereberal (IC), intraorgan (e.g. intrahepatic), slow release
implant, or subcutaneous administration, or via administration
using an osmotic or mechanical pump or via inhalation. Preparation
of formulations for delivery via such methods, and delivery using
such methods, are well known in the art.
[0051] The complexes can be optimized for target cell type through
the choice and ratio of lipids, the ratio of ligand (e.g.,
protein/peptide, antibody or antibody fragment) to liposome, the
ratio of ligand and liposome to the therapeutic agent, and the
choice of ligand and therapeutic agent.
[0052] The complexes made in accordance with the methods of this
invention can be provided in the form of kits for use in the
systemic delivery of a nucleic acid, therapeutic molecule, or other
payload by the complex. Suitable kits can comprise, in separate,
suitable containers, the liposome, the ligand (e.g.,
protein/peptide, antibody or antibody fragment), and the nucleic
acid, the therapeutic or diagnostic agent. The components can be
mixed under sterile conditions in the appropriate order and
administered to a patient within a reasonable period of time,
generally from about 30 minutes to about 24 hours, after
preparation. The kit components preferably are provided as
solutions or as dried powders. Components provided in solution
form, preferably are formulated in sterile water-for-injection,
along with appropriate buffers, osmolarity control agents, etc. The
complete complex can also be formulated as a dried powder
(lyophilized) (see, e.g., U.S. Published Patent Application No.
2005/0002998, the disclosure of which is incorporated by reference
herein in its entirety).
[0053] As discussed throughout U.S. Published Patent Application
Nos. 2003/0044407 and 2007/0065499, the disclosures of which are
incorporated herein by reference, the cationic liposome complexes
described throughout have successfully delivered various
therapeutic and diagnostic agents to tumor cells, via targeting
using the anti-transferring single chain antibody fragment
(TfRscFv). Specifically, nucleic acid molecules, e.g., antisense
and siRNA, as well as plasmid DNA (such as p53 and RB94), have been
successfully delivered using the liposome complexes of the present
invention (see U.S. Published Patent Application Nos. 2003/0044407
and 2007/0065499).
[0054] Cationic Liposome Complexes for Prevention or Treatment
Toxicity Associated with Organophosphate Agents
[0055] As described above, the use of BChE as an OP bioscavenger
can be used as a prophylactic agent. However, the inability to
produce and rapidly deliver sufficient quantities when needed is a
major hurdle to implementation of these use of this compound.
Therefore, it is critical to develop a means to increase expression
of BChE in the circulation and in other tissues such as lung, liver
and brain.
[0056] Recombinant human BChE has been produced using mammalian
cell cultures, transgenic goats, plants and silk worm larvae (23).
Although this recombinant BChE proved to be successful for
protection against lethal doses of soman and VX, production through
these methods is not practical given the high cost of producing
large quantities of GMP material and potential stability and safety
issues. Thus, other approaches are being explored. Parikh and
colleagues (23) have used adenovirus to deliver mouse BChE. They
found that this intravenously administered Ad-MoBChE was able to
protect mice from multi LD.sub.50 doses of OPs VX and echothiophate
at a level equivalent to a multimilligram injection of pure BChE.
Although elevated amounts of BChE were produced, the levels
achieved did not translate to production of the amount of BChE
required to protect a human, estimated at a minimum to be 250 mg/70
kg. One possible reason for this is that the BChE protein produced
in the mice was not the tetrameric form, but was primarily dimers
(85%) which are eliminated rapidly in mammals. The therapeutic
potential of BChE is dependent on its ability to remain in
circulation for an extended period of time. This stability is in
turn dependent on maintaining the tetrameric structure. Moreover,
the use of viral vectors for delivery systems have potential
immunogenicity problems and drawbacks associated with the necessity
for intravenous administration and repeat administration.
[0057] Thus, other methods need to be developed that do not have
immunogenicity issues associated with repeat administration; can be
easily self administered in the field; and, more importantly, can
result in significantly high levels of expression of the preferred
tetrameric form of BChE with increased time in circulation. The
liposome approach described herein offers a number of advantages
over viral methodologies for gene delivery including lack of
immunogenicity (24). Cationic liposomes have proven to be safe and
efficient for in vivo gene delivery (25,26). More than 110 clinical
trials using cationic liposomes for DNA delivery, including 85 in
the US, have been approved (27) and at least 6 liposome-based
products are on the market (28).
[0058] As described herein, targeted liposome complexes carrying
both a plasmid encoding BChE, including mutant forms such as the
more active G117H mutant form (see e.g., Reference 11, the
disclosure of which is incorporated by reference here in its
entirety) of BChE, and a plasmid encoding the gene for a proline
rich peptide (such as rQ45) to facilitate tetramerization, are
provided. When administered at the threat of potential OP exposure,
the genes are efficiently delivered to various tissues including
lung, brain and liver, with subsequent high levels of expression of
fresh, OP resistant, tetrameric hBChE, being continuously produced
and present in the circulation for an extended period of time
(e.g., 1 Day to 14 days or longer). As described herein, such
methods are suitably a prophylactic measure designed to protect if
there is a possibility of nerve gas exposure, and as a
post-exposure therapeutic OP countermeasure. It has been
established that genetic variants of human BChE which can increase
the sensitivity of individuals to OPs are common (29,30). The
methods described herein can provide high levels of exogenous,
active BChE to override this inherent genetic sensitivity and
protect both this subset and the general population.
Preparation of Liposome Complexes
[0059] In an exemplary embodiment, the present invention provides
methods of preparing a ligand-targeted (e.g., protein/peptide,
antibody- or antibody fragment-targeted) cationic liposome complex.
In suitable embodiments, such methods comprise preparing a ligand
(e.g., protein/peptide, antibody or antibody fragment), and mixing
the ligand with a cationic liposome to form a ligand-targeted
cationic liposome, wherein the ligand is directly
associated/complexed with, but is not chemically conjugated to, the
cationic liposome. The ligand-targeted cationic liposome is then
mixed with one or more nucleic acid molecules.
[0060] In embodiments the nucleic acid molecules comprise a nucleic
acid molecule encoding butyrylcholinesterase (BChE) associated with
the cationic liposome and a nucleic acid molecule encoding a
polyproline rich peptide, associated with the cationic liposome, to
form the ligand-targeted cationic liposome complex. Sequences of
nucleic acid molecules encoding BChE are readily known in the art
or otherwise described herein. See, e.g., McTiernan, et al., "Brain
cDNA clone for human cholinesterase," Proc. Natl. Acad. Sci.
84:6682-6686, FIG. 2, the disclosure of which is incorporated by
reference herein in its entirety for all purposes.
[0061] Sequences of nucleic acid molecules encoding a polyproline
rich peptide are described herein or otherwise known in the art. An
exemplary nucleic acid encoding the amino terminal 45 residues
(rQ45) of the rat collagen tail, including the polyproline rich
peptide domain (also called proline-rich attachment domain (PRAD))
are disclosed in, for example, Altamirano and Lockridge,
Chemico-Biological Interactions 119-120:53-60 (1999) (see page 57,
section 2.9); Krejci et al., The Journal of Biological Chemistry
272:22840-22847 (1997) (see page 22842, FIG. 1); and Bon et al.,
The Journal of Biological Chemistry 272:3016-3021 (1997) (see
section bridging pages 3016-3017) (the disclosures of each of which
are incorporated by reference herein in their entireties).
[0062] In another exemplary embodiment, the present invention
provides methods of preparing a ligand-targeted (e.g.,
protein/peptide, antibody- or antibody fragment-targeted) cationic
liposome complex. In suitable embodiments, such methods comprise
preparing a ligand (e.g., protein/peptide, antibody or antibody
fragment), and mixing the ligand with a cationic liposome to form a
ligand-targeted cationic liposome, wherein the ligand is directly
complexed with the cationic liposome via chemical conjugation. The
ligand-targeted cationic liposome is then mixed with one or more
nucleic acid molecules as described herein, to form the
ligand-targeted cationic liposome complex.
[0063] As described herein, nucleic acid molecules are associated
with the cationic liposome, which includes the nucleic acid
molecules being suitably encapsulated, contained or
complexed/associated with the liposome complexes of the present
invention by simply mixing the one or more nucleic acid molecules
with the liposomes during processing. Suitable ratios of nucleic
acid molecules:liposome complexes are readily determined by the
ordinarily skilled artisan and described herein.
[0064] In embodiments, the nucleic acid molecule encoding BChE is
contained in a first plasmid and the nucleic acid molecule encoding
the polyproline rich peptide is contained in a second plasmid. That
is, the proteins are encoded from different plasmids. However, in
further embodiments, the proteins can be encoded from the same
plasmid, i.e., both genes contained in the same plasmid.
[0065] Suitably, the two distinct plasmids utilized to encode the
nucleic acid molecules are the same, with the exception of the gene
that they encode. In suitable embodiments the nucleic acid molecule
encoding BChE, and the nucleic acid molecule encoding the
polyproline rich peptide, are placed downstream from a nucleic acid
promoter, for example, a promoter as described in U.S. Published
patent application No. 2007/0065432, the disclosure of which is
incorporated by reference herein in its entirety. Suitably, the
nucleic acid molecules are placed in a plasmid construct,
comprising, from 5' to 3': (a) at least one human adenovirus
enhancer sequence; (b) a cytomegalovirus (CMV) promoter; (c) a
multiple cloning site; (d) the nucleic acid molecule encoding BChE
of the nucleic acid molecule encoding the polyproline rich peptide;
and (e) an SV 40 poly A sequence, wherein the 3' end of the plasmid
construct does not comprise adenovirus map units 9-16 when compared
to a wild-type adenovirus. See FIG. 3 of U.S. Published Patent
Application No. 2007/0065432, which is specifically incorporated by
reference herein.
[0066] Alternatively the nucleic acid molecule encoding BChE, and
the nucleic acid molecule encoding the polyproline rich peptide,
are in the same construct, but the nucleic acid molecule encoding
BChE is placed downstream from the high expression promoter
disclosed in U.S. Published Patent Application No. 2007/0065432,
while the nucleic acid molecule encoding the polyproline rich
peptide is placed downstream of a standard promoter such as RSV or
CMV.
[0067] In exemplary embodiments, the nucleic acids are present at a
molar ratio of about 10:01 to about 0.1:10 (moles nucleic acid
molecule encoding butyrylcholinesterase (BChE):moles nucleic acid
molecule encoding a polyproline rich peptide). In exemplary
embodiments, the nucleic acids are present at a molar ratio of
about 10:1 to about 1:10 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide). For example, the nucleic acid molecules
encoding butyrylcholinesterase (BChE) and the nucleic acid molecule
encoding a polyproline rich peptide are present in the liposomes at
a molar ratio in the range of about 10:1 to about 1:10, or about
5:1 to about 1:5, or more suitably about 4:1 (moles nucleic acid
molecule encoding butyrylcholinesterase (BChE):moles nucleic acid
molecule encoding a polyproline rich peptide), or more suitably the
nucleic acid molecules are present at a molar ratio of about 2:1
(moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide) or more suitably the nucleic acid molecules are present at
a molar ratio of about 1:1 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide). Additional molar ratios outside of these
ranges can also be used.
[0068] Suitably, the molar ratio of nucleic acid molecules to
liposome complex is in the range of about 0.5:1 to about 1:40
(.mu.g total nucleic acid:.mu.g liposome), or about 1:1 to about
1:40 (.mu.g total nucleic acid:.mu.g liposome), suitably about 1:5
to about 1:20 (.mu.g total nucleic acid:.mu.g liposome), more
suitably about 1:10 (.mu.g total nucleic acid:.mu.g liposome). As
utilized herein, the molar ratio of nucleic acid molecules to
liposome complex includes both "populations" of nucleic acids,
i.e., the total amount of nucleic acid includes one or more nucleic
acid molecules encoding BChE, and one or more nucleic acid
molecules encoding a polyproline rich peptide.
[0069] As described throughout, examples of desirable cationic
liposomes for delivery of nucleic acid molecules include those that
comprise a mixture of dioleoyltrimethylammonium phosphate (DOTAP)
and dioleoylphosphatidylethanolamine (DOPE) and/or cholesterol
(chol); and a mixture of dimethyldioctadecylammonium bromide (DDAB)
and DOPE and/or chol. The ratio of the lipids can be varied to
optimize the efficiency of uptake of the nucleic acid molecules for
the specific target cell type. The liposome can comprise a mixture
of one or more cationic lipids and one or more neutral or helper
lipids. A desirable ratio of cationic lipid(s) to neutral or helper
lipid(s) is about 1:(0.5-3), preferably about 1:(1-2) (molar
ratio). Examples of ratios of various lipids useful in the practice
of the present invention include, but are not limited to:
TABLE-US-00001 LipA DOTAP/DOPE 1:1 molar ratio LipB DDAB/DOPE 1:1
molar ratio LipC DDAB/DOPE 1:2 molar ratio LipD DOTAP/Chol 1:1
molar ratio LipE DDAB/Chol 1:1 molar ratio LipG DOTAP/DOPE/Chol
2:1:1 molar ratio LipH DDAB/DOPE/Chol 2:1:1 molar ratio (DOTAP =
dioleoyltrimethylaminnonium phosphate, DDAB =
dimethyldioctadecylammonium bromide; DOPE =
dioleoylphosphatidylethanolamine; chol = cholesterol).
[0070] As discussed throughout, in suitable embodiments, the ligand
used in the compositions and methods of the present invention is an
antibody fragment, for example a single chain Fv fragment, such as,
an anti-transferrin receptor single chain Fv (TfRscFv). Suitably,
the antibody or antibody fragment is mixed with the cationic
liposome at a ratio in the range of about 1:1 to about 1:100,
suitably about 1:10 to about 1:50 (w:w), more suitably about 1:30,
or about 1:33 (antibody or antibody fragment:lipid) to form the
targeted cationic liposome. In additional embodiments, the
liposomes can also comprise endosomal disrupting peptides, such as
the K[K(H)KKK].sub.5-K(H)KKC (HoKC) (HK) (SEQ ID NO: 1) peptide
manufactured by Sigma-Genosys (The Woodlands, Tex.), associated
with the liposomes. The endosomal disrupting peptide HoKC may help
the release of agents in the cytoplasm of the cells.
[0071] In exemplary embodiments, cationic liposomal formulations A
(DOTAP:DOPE at a 1:1 molar ratio), B (DDAB:DOPE at 1:1), G
(DOTAP:DOPE:cholesterol at 1:1:1) and H (DDAB:DOPE:cholesterol at
1:1:1) or any of the lipid formulations described throughout, are
prepared using the ethanol injection method as described herein and
in the Examples. Only when a peptide (such as the
K[K(H)KKK].sub.5-K(H)KKC (HoKC) (HK) peptide) is included in the
complex, each liposome formulation also suitably includes MPB-DOPE
at 5 molar percent of total lipid. Complexes prepared without the
peptide do NOT contain MPB-DOPE and thus a ligand cannot be bound
to the liposome via chemical conjugation.
[0072] Since the HoKC peptide (K[K(H)KKK].sub.5-K(H)KKC) (SEQ ID
NO: 1) carries a terminal cysteine, MPB-DOPE was included in all of
the liposome compositions to allow conjugation of peptide to the
liposome. The Lip-HoKC liposomes were prepared using the coupling
reaction between the cationic liposomes carrying the maleimide
group (Lip-MPB) and the peptide. An aliquot of 0.1 mMol of the
peptide with a free thiol group on cysteine was added to 2 mMol of
Lip-MPB in 10 mM HEPES, pH 7.4, solution and rotated at room
temperature (20-30 r.p.m.) for 2 h. The resulting Lip-HoKC has a
lipid concentration of 1.4 mM.
[0073] The full complex is formed in a manner identical to that
used to produce the TfRscFv:Lip:DNA complex without HoKC. Here also
the anti-transferrin receptor single chain antibody fragment
(TfRscFv) is mixed with Lip-HoKC by gentle inversion at a specific
ratio, and incubated at room temperature for 10 min. Nucleic acid
is then added to the TfRscFv:Lip-HoKC solution, mixed by gentle
inversion and again incubated at room temperature for 15 min, after
which dextrose or sucrose is added to a final concentration of
5-10% and again mixed by gentle inversion. A suitable ratio for the
TfRscFv:Lip-HoKC:Nucleic acid complex is 0.3 mg:7 nmol:1 mg.
[0074] In additional embodiments, the methods of the present
invention comprise preparing a ligand (e.g., protein/peptide,
antibody or antibody fragment), and chemically conjugating the
ligand to the surface of a cationic liposome to form a cationic
immunoliposome. The cationic liposome is then mixed with a plasmid
construct (vector) encoding BChE, and a plasmid construct (vector)
encoding a polyproline rich peptide, to form the ligand-targeted
liposome complex. Exemplary methods, composition, ratios and
conditions for chemically conjugating ligands (e.g.,
proteins/peptides, antibodies or antibody fragments) to cationic
liposomes are disclosed in U.S. Pat. No. 7,479,276, the disclosure
of which is incorporated by reference herein in its entirety.
[0075] As used herein the terms "protein" "peptide" and
"polypeptide" are used interchangeably to mean any chain or chains
of two or more amino acids, and does not refer to a specific length
of product. Thus, peptides, dipeptides, tripeptides, oligopeptides,
"protein," "amino acid chain," or any other term used to refer to a
chain or chains of two or more amino acids, are included within the
definition of the terms "protein," "polypeptide," and
"peptide."
[0076] The present invention also provides cationic liposome
complexes prepared according to the methods described throughout.
For example, the present invention provides ligand-targeted (e.g.,
protein/peptide, antibody- or antibody fragment-targeted) cationic
liposome complexes comprising a cationic liposome, a ligand (e.g.,
protein/peptide, antibody or antibody fragment), one or more
nucleic acid molecules encoding BChE, and one or more nucleic acid
molecules encoding a polyproline rich peptide, wherein the ligand
is directly complexed/associated with, but not chemically
conjugated to the cationic liposome. The ligand (e.g.,
protein/peptide, antibody or antibody fragment) is suitably
associated with the liposome via an interaction (e.g.,
electrostatic, van der Walls, or other non-chemically conjugated
interaction) between the ligand and the liposome. In general, a
linker or spacer molecule (e.g., a polymer or other molecule) is
not used to attach the ligands and the liposome when non-chemically
conjugated.
[0077] As described herein, in additional embodiments, the ligand
(e.g., protein/peptide, antibody or antibody fragment) is
chemically conjugated to the cationic liposomes, for example, via a
chemical interaction between the cationic liposome which contains a
maleimidyl group or other sulfhydryl-reacting group, and a sulfur
atom on the ligand (e.g., protein/peptide, antibody or antibody
fragment). The nucleic acids are then added to the liposome to form
the liposome-DNA complexes, or the nucleic acids can be added
first, and then complexed with the ligands. Such methods are
disclosed in U.S. Pat. No. 7,479,276, the disclosure of which is
incorporated by reference herein in its entirety.
[0078] The nucleic acid molecules can be encapsulated within the
cationic liposome, contained within a hydrocarbon chain region of
the cationic liposome, associated with an inner or outer monolayer
of the cationic liposome (e.g., the head-group region), or any
combination thereof. Suitably, the cationic liposomes of the
present invention are unilamellar liposomes (i.e. a single
bilayer), though multilamellar liposomes which comprise several
concentric bilayers can also be used. Single bilayer cationic
liposomes of the present invention comprise an interior aqueous
volume in which nucleic acid molecules can be encapsulated. They
also comprise a single bilayer which has a hydrocarbon chain region
(i.e., the lipid chain region of the lipids) in which nucleic acid
molecules that have been conditioned to be neutral or largely
uncharged can be contained. In addition, nucleic acid molecules can
be complexed or associated with either, or both, the inner
monolayer and/or the outer monolayer of the liposome membrane
(i.e., the head-group region of the lipids), e.g., via a
charge-charge interaction between the negatively charged nucleic
acid molecules and the positively charged cationic liposomes. In
further embodiments, nucleic acid molecules can be
encapsulated/associated/complexed in any or all of these regions of
the cationic liposome complexes of the present invention.
[0079] As discussed throughout, suitably the nucleic acid molecules
are present at a molar ratio of about 0.1 mole of one or more
nucleic acid molecules encoding butyrylcholinesterase (BChE), to
about 10 mole of one or more nucleic acid molecules encoding a
polyproline rich peptide; to 10 mole of one or more nucleic acid
molecules encoding butyrylcholinesterase (BChE), to about 0.1 mole
of one or more nucleic acid molecules encoding a polyproline rich
peptide; in further embodiments the nucleic acid molecules are
present at a molar ratio of about 1 mole of one or more nucleic
acid molecules encoding butyrylcholinesterase (BChE), to about 10
mole of one or more nucleic acid molecules encoding a polyproline
rich peptide; to 10 mole of one or more nucleic acid molecules
encoding butyrylcholinesterase (BChE), to about 1 mole of one or
more nucleic acid molecules encoding a polyproline rich peptidemore
suitably the nucleic acid molecules are present at a molar ratio of
about 5 mole of one or more nucleic acid molecules encoding
butyrylcholinesterase (BChE), to about 1 mole of one or more
nucleic acid molecules encoding a polyproline rich peptide; to 1
mole of one or more nucleic acid molecules encoding
butyrylcholinesterase (BChE), to about 5 mole of one or more
nucleic acid molecules encoding a polyproline rich peptide; in
particular, about 4 mole of one or more nucleic acid molecules
encoding butyrylcholinesterase (BChE), to about 1 mole of one or
more nucleic acid molecules encoding a polyproline rich peptide, in
particular, about 2 mole of one or more nucleic acid molecules
encoding butyrylcholinesterase (BChE), to about 1 mole of one or
more nucleic acid molecules encoding a polyproline rich peptide, in
particular, about 1 mole of one or more nucleic acid molecules
encoding butyrylcholinesterase (BChE), to about 1 mole of one or
more nucleic acid molecules encoding a polyproline rich peptide can
be used in the liposome complexes. Suitable amounts/ratios of
lipids, targeting ligands and nucleic acid molecules are also
described throughout.
[0080] In exemplary embodiments, the one or more nucleic acid
molecules encoding butyrylcholinesterase (BChE), the one or more
nucleic acid molecules encoding a polyproline rich peptide, and the
liposomes, are present at a weight ratio of between about 0.5:1 to
about 1:40 (.mu.g total nucleic acid:.mu.g lipid), suitably about
1:1 to about 1:40 (.mu.g total nucleic acid:.mu.g lipid), or about
1:5 to about 1:20 (.mu.g total nucleic acid:.mu.g lipid), e.g.,
about 1:10 (.mu.g total nucleic acid:.mu.g lipid). An exemplary
liposome composition of the present invention comprises total
nucleic acid, lipid and a ligand, such as a single chain antibody
(e.g., TfscFv) at a weight ratio of about 1:10:0.33 (.mu.g total
nucleic acid:.mu.g lipid:.mu.g single chain antibody) (also
referred to herein as scL).
[0081] The present invention also provides pharmaceutical
compositions comprising the ligand-targeted cationic liposome
complexes described throughout. In suitable embodiments, the
pharmaceutical compositions further comprise one or more excipients
selected from the group consisting of one or more antibacterials
(e.g., amphotericin B, chloretracycline, gentamicin, neomycin), one
or more preservatives (e.g., benzethonium chloride, EDTA,
formaldehyde, 2-phenoxyethanol), one or more buffers (e.g.,
phosphate buffers, sodium borate, sodium chloride), one or more
surfactants (polysorbate 20, 80), one or more protein stabilizers
(e.g., albumin, lactose, potassium glutamate), sugars e.g. sucrose
or dextrose, and adjuvants (e.g., aluminum hydroxide, aluminum
phosphate). Additional excipients are well known in the art and can
be readily used in the practice of the present invention.
[0082] In certain such embodiments, the nucleic acid molecules
encoding butyrylcholinesterase (BChE) are delivered in the same
composition as the nucleic acids encoding a polyproline rich
peptide, but rather than being encapsulated/associated with the
same cationic liposome, they are associated with two (or more)
different cationic liposomes and then delivered in the same
composition (i.e., two different liposome populations mixed
together). In other embodiments the nucleic acid molecules encoding
BChE and the nucleic acid molecules encoding a polyproline rich
peptide can be contained together within the same plasmid vector.
The present invention also encompasses administering to a mammal
two separate liposome compositions, one which comprises nucleic
acids encoding BChE, and one which comprises nucleic acids encoding
a polyproline rich peptide. Examples of suitable lipids, targeting
molecules and nucleic acid molecules, and ratios of such components
for use the pharmaceutical compositions of the present invention
are described throughout.
Methods of Treatment and/or Prevention
[0083] Also provided herein are methods of treating and/or
preventing toxicity associated with exposure to an organophosphate
agent in a mammal. Suitably such methods comprise administering to
the mammal a cationic liposome complex, wherein the cationic
liposome complex comprises a cationic liposome, a ligand directly
complexed with, but not chemically conjugated to, the cationic
liposome, a nucleic acid molecule (including one or more than one
nucleic acid molecules) encoding butyrylcholinesterase (BChE)
associated with the cationic liposome, and a nucleic acid molecule
(including one or more than one nucleic acid molecules) encoding a
polyproline rich peptide associated with the same or a different
cationic liposome.
[0084] The methods described herein can be utilized with any
mammal, including humans, dogs, cats, mice, rats, monkeys, etc.
[0085] Suitably, the methods are for the treatment of toxicity
associated with exposure to the organophosphate agent. As used
herein, methods "for treatment" of the toxicity associated with
exposure to an OP agent provide a therapeutically effective amount
of BChE and a polyproline rich peptide so as to allow the mammal to
overcome the toxicity associated with exposure to an OP agent,
after the mammal has been exposed to the OP agent.
[0086] In further embodiments, the methods are for the prevention
of toxicity associated with exposure to the organophosphate agent.
As used herein, methods "for prevention" of the toxicity associated
with exposure to an OP agent provide a therapeutically effective
amount of BChE and a polyproline rich peptide before (i.e., if) the
mammal is exposed to the OP agent, so as to allow the mammal to
overcome the toxicity associated with a future exposure to an OP
agent.
[0087] The phrase "therapeutically effective amount" is used here
to mean an amount sufficient to reduce by at least about 10
percent, suitably at by at least 20 percent, or by at least 30
percent, or by at least 40 percent, more suitably by at least 50-90
percent, and still more suitably prevent (i.e. reduce by 100
percent), a clinically significant deficit in the activity,
function and response of the mammal to the toxicity associated with
the OP agent. In addition, a "therapeutically effective amount" is
suitably also sufficient to cause an improvement in a clinically
significant condition in the mammal.
[0088] In the context of detoxifying agents (i.e., BChE), often the
level of effectiveness is defined in terms of
protecting/scavenging/detoxifying a certain amount of toxic agent
(OP). A useful metric is "LD.sub.50," which is the amount of
chemical that is lethal to one half of the animals exposed to the
chemical agent. Thus, a "therapeutically effective amount" is also
an amount that is sufficient to protect against at least one
LD.sub.50 (i.e., 1.times.LD.sub.50--1 times LD.sub.50) of the toxic
agent (OP). As described herein, the methods provided suitably
deliver amounts of detoxifying agent that can treat the effects of
exposure to, or prevent the potential exposure to, at least 1, at
least 2, at least 3, at least 4, at least 5, at least 6 times (or
more) the LD.sub.50 of the OP agent.
[0089] Suitably the complex is administered so as to treat toxicity
associated with exposure to at least 1.times.LD.sub.50 of the
organophosphate agent, up to and including, 5.times.LD.sub.50 of
the organophosphate agent.
[0090] In other embodiments, the complex is administered so as to
prevent toxicity associated with the potential exposure to at least
1.times.LD.sub.50 of the organophosphate agent, up to and including
5.times.LD.sub.50 of the organophosphate agent.
[0091] Any suitable method can be used to administer the complexes
as described herein to the mammal. In embodiments, the complex is
administered via a route selected from the group consisting of
intranasal administration, intravenous administration, oral
administration, sublingual administration, intramuscular
administration, intralesional administration, intradermal
administration, transdermal administration, intraocular
administration, intraperitoneal administration, percutaneous
administration, aerosol administration, intraorgan administration,
intracereberal administration, topical administration, subcutaneous
administration, endoscopic administration, slow release implant,
administration via an osmotic or mechanical pump and administration
via inhalation. Most suitably, the complex is administered via
intranasal or aerosol inhalation administration, resulting in
pulmonary delivery of the complexes and nucleic acids.
[0092] Pulmonary delivery devices for administration of the
complexes described herein are well known in the art. Pulmonary
delivery devices generate particles of active agent, typically
about 0.01 .mu.m to about 4 .mu.m, which may be inhaled by the
subject. Pulmonary delivery devices are widely used for inhalation
of an active agent (i.e., the liposome complexes described herein)
from solution or suspension, or inhalation of an active agent in
dry powder form, optionally admixed with an excipient. Examples of
pulmonary delivery devices include, but are not limited to, metered
dose inhalers (MDIs), nebulizers, and dry powder inhalers (DPIs).
The pulmonary delivery devices may optionally be pressurized, and
may utilize propellant systems. The pulmonary delivery devices may
also incorporate holding chambers, e.g., spacers, to prevent
aerosolized active agents from escaping into the air, and allowing
the subject more time to inhale.
[0093] Pulmonary delivery devices utilized in the methods described
herein may be activatable by inhalation, e.g., will automatically
dispense active agent upon inhalation by the subject, and may be
used with aerosol containers which contain active agents and
optionally contain propellants. These devices can administer a
plurality of metered doses in a controlled manner, allowing
controlled and consistent dosing of active agents into the
subject's bronchial passages and pulmonary epithelium. Examples of
pulmonary delivery devices are described in U.S. Pat. Nos.
5,290,539, 6,615,826, 4,349,945, 6,460,537, 6,029,661, 5,672,581,
5,586,550, and 5,511,540, which are incorporated by reference
herein.
[0094] Metered Dose Inhalers (MDIs) operate by utilizing a
propellant to eject a constant volume of an active agent, which is
inhaled by the subject. MDIs may also include a surfactant to
prevent aggregation of the active agent. The active agent may be
dissolved or suspended in solution. MDIs utilizing propellants may
require simultaneous inhalation and activation of the MDI. Holding
chambers, e.g., spacers, may be used to store the aerosolized
active agent, eliminating the need for simultaneous activation and
inhalation. MDIs provide a constant, metered dosage of the active
agent and allow for consistent dosing. Examples of MDIs are
described in U.S. Pat. Nos. 6,615,826 and 5,290,539.
[0095] Nebulizers operate by creating a mist, i.e., nebulizing or
atomizing, a formulation of active agent in solution, which is
inhaled by the subject. The active agent may be dissolved or
suspended in solution. The droplets may be created by any method
known in the art, including the use of a fan, a vibrating member,
or ultrasonic apparatus. Nebulizers are more gentle than MDIs and
DPIs, and are appropriate for individuals unable to use inhalers,
such as infants, young children, and individuals that are seriously
ill or incapacitated. Examples of nebulizers are described in U.S.
Pat. Nos. 6,748,945, 6,530,370, 6,598,602, and 6,009,869.
[0096] Dry Powder Inhalers (DPIs) do not use propellants, and
administer dry powder which is inhaled by the subject. To
distribute the dry powder, DPIs may utilize any method known in the
art to propel the active agent, including pneumatic systems,
powered fans, or mechanical propulsion, e.g., squeezing of the
container. DPIs may instead rely simply upon the inhalation by the
subject. Blending of active agent with propellants is not required
for DPIs, allowing delivery of larger payloads of active agent. An
example of a DPI is described in U.S. Pat. No. 6,029,661.
[0097] Often, the aerosolization of a liquid or a dry powder
formulation for inhalation into the lung will require a propellant.
The propellant may be any propellant generally used in the art.
Specific non-limiting examples of such useful propellants are a
chlorotlourocarbon, a hydrofluorocarbon, a hydochlorofluorocarbon,
or a hydrocarbon, including triflouromethane,
dichlorodiflouromethane, dichlorotetrafiioroethanol, and
1,1,1,2-tetraflouroethane, or combinations thereof. Examples of
propellant formulations are described in U.S. Pat. No. 5,672,581,
which is incorporated herein by reference.
[0098] Other methods known in the art for pulmonary administration
can also be used. By way of example, these methods include delivery
by intratracheal inhalation, insufflation, or intubation, e.g., the
delivery of a solution, a powder, or a mist into the lungs by a
syringe, tube, or similar device.
[0099] As described herein, suitably the ligand for use in the
complexes is transferrin, an antibody and an antibody fragment.
Exemplary antibodies and antibody fragments are described
throughout and include single chain Fv antibody fragment, such as
an anti-transferrin receptor single chain Fv (TfRscFv).
[0100] In embodiments, the ligand-targeted cationic liposomes
further comprise a peptide comprising a K[K(H)KKK]5-K(H)KKC (HoKC)
(SEQ ID NO: 1) peptide associated with the cationic liposome.
Exemplary methods of associating the peptide with the cationic
liposomes are described herein.
[0101] In suitable embodiments, the nucleic acid molecule encoding
BChE is contained in a first plasmid and the nucleic acid molecule
encoding the polyproline rich peptide is contained in a second
plasmid. That is, the nucleic acid molecules are contained in
separate plasmids, each of which plasmids are associated (i.e.,
encapsulated) with the same or different cationic liposomes. In
further embodiments, the nucleic acid molecule encoding BChE and
the nucleic acid molecule encoding the polyproline rich peptide can
be contained in the same plasmid, i.e., expressed from the same
plasmid. However, by utilizing two separate plasmids, the amount of
each nucleic acid, and thus the amount of each expressed protein,
can be controlled separately and the ratios of the nucleic acids
optimized for various treatment or prevention protocols and
effects.
[0102] Suitably, the nucleic acid molecule encoding BChE is
contained in a first plasmid construct, comprising, from 5' to 3':
(a) at least one human adenovirus enhancer sequence; (b) a
cytomegalovirus (CMV) promoter; (c) a multiple cloning site; (d)
the nucleic acid molecule encoding BChE; and (e) an SV 40 poly A
sequence, wherein the 3' end of the plasmid construct does not
comprise adenovirus map units 9-16 when compared to a wild-type
adenovirus. In embodiments, the nucleic acid molecule encoding the
polyproline rich peptide is contained in a second plasmid
construct, comprising, from 5' to 3': (a) at least one human
adenovirus enhancer sequence; (b) a cytomegalovirus (CMV) promoter;
(c) a multiple cloning site; (d) the nucleic acid molecule encoding
the polyproline rich peptide; and (e) an SV 40 poly A sequence,
wherein the 3' end of the plasmid construct does not comprise
adenovirus map units 9-16 when compared to a wild-type adenovirus.
See FIG. 3 of U.S. Published Patent Application No. 2007/0065432,
which is specifically incorporated by reference herein, which
describes such constructs. In further embodiments, the nucleic
acids can be expressed by other promoters known in the art.
[0103] In exemplary embodiments, the BChE that is expressed by the
nucleic acids is a mutant version of BChE. Suitable mutants of BChE
are known in the art or otherwise described herein, and suitably
include the G117H mutant, as described in Millard, C. B.,
Lockridge, O., & Broomfield, C. A. (1995), "Design and
expression of organophosphorus acid anhydride hydrolase activity in
human butyrylcholinesterase," Biochemistry 34: 15925-15933; and
Lockridge, O., Blong, R. M., Masson, P., Froment, M. T., Millard,
C. B., & Broomfield, C. A. (1997), "A single amino acid
substitution, Gly117His, confers phosphotriesterase
(organophosphorus acid anhydride hydrolase) activity on human
butyrylcholinesterase," Biochemistry 36:786-795, the disclosures of
each of which are incorporated by reference herein in their
entireties.
[0104] As described herein, suitably the cationic liposome
comprises a mixture of one or more cationic lipids and one or more
neutral or helper lipids. Exemplary ratios of ligand:cationic
liposome are described throughout, and suitably include where the
ligand and the cationic liposome are present at a ratio in the
range of about 1:1 to about 1:100 (w:w), suitably where the ligand
and the cationic liposome are present at a ratio in the range of
about 1:10 to about 1:50 (w:w), or more suitably where the ligand
and the cationic liposome are present at a ratio in the range of
about 1:20 to about 1:40 (w:w).
[0105] In embodiments, the cationic liposomes for use in the
methods described herein comprise a mixture of
dioleoyltrimethylammonium phosphate with
dioleoylphosphatidylethanolamine and cholesterol; a mixture of
dioleoyltrimethylammonium phosphate with cholesterol, a mixture of
dimethyldioctadecylammonium bromide with
dioleoylphosphatidylethanolamine and cholesterol, a mixture of
dimethyldioctadecylammonium bromide with
dioleoylphosphatidylethanolamine, a mixture of
dimethyldioctadecylammonium bromide with cholesterol, or a mixture
of dioleoyltrimethylammonium phosphate with
dioleoylphosphatidylethanolamine. Additional liposome compositions
that can be utilized in the methods are described herein.
[0106] In suitable embodiments, the nucleic acid molecules are
present at a molar ratio of about 10:1 to about 1:10 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE): moles nucleic
acid molecule encoding a polyproline rich peptide). More suitably,
the nucleic acid molecules are present at a molar ratio of about
8:1 to about 1:8 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide), or a molar ratio of about 7:1 to about
1:7 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide), a molar ratio of about 6:1 to about 1:6 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding a polyproline rich peptide) a molar ratio of
about 5:1 to about 1:5 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide), a molar ratio of about 4:1 to about 1:4
(moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide), a molar ratio of about 3:1 to about 1:3 (moles nucleic
acid molecule encoding butyrylcholinesterase (BChE):moles nucleic
acid molecule encoding a polyproline rich peptide), a molar ratio
of about 2:1 to about 1:2 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide), or a molar ratio of about 1:1 (moles
nucleic acid molecule encoding butyrylcholinesterase (BChE):moles
nucleic acid molecule encoding a polyproline rich peptide).
[0107] Suitable ratios for the total amount of nucleic acid (i.e.
the amount nucleic acid molecules encoding butyrylcholinesterase
(BChE) combined with the nucleic acid molecules encoding a
polyproline rich peptide) to the amount of liposome are described
herein. In suitable embodiments, the weight ratio is between about
1:1 to about 1:40 (.mu.g nucleic acid:.mu.g liposome), more
suitably the weight ratio is between about 1:5 to about 1:20 (.mu.g
total nucleic acid:.mu.g liposome), or the weight ratio is about
1:10 (.mu.g total nucleic acid:.mu.g liposome).
[0108] Suitably, the nucleic acid molecules are encapsulated within
the interior of the cationic liposome, though in other embodiments
the nucleic acid molecules can be associated with an inner or outer
monolayer of the cationic liposome (e.g., the head-group
region).
[0109] In suitable embodiments of the methods of treating toxicity
associated with exposure to an OP agent, the complexes described
herein are administered immediately after exposure to the
organophosphate agent, i.e., within seconds (10, 20, 30, seconds,
etc.) minutes (1, 5, 10, 15, 20, or 30 or more minutes), hours
(i.e., 1, 2, 3, 4, 5 or more hours) or days (i.e., 1, 2, 3, 4, 5 or
more days) after the mammal is contacted with the OP agent. In
general, such contact with the OP agent will be via inhalation of
the agent and/or contact with the skin, eyes or mucus membranes of
the mammal.
[0110] In suitable embodiments of the methods of preventing
toxicity associated with exposure to an OP agent, the complexes
described herein are administered at least 6 hours prior to a
potential exposure to the organophosphate agent. Suitably, the
complexes described herein are administered at least 10 hours, at
least 12 hours, at least 15 hours, at least 20 hours, at least 24
hours, at least 36 hours or at least 48 hours prior to the
potential exposure to the organophosphate agent. In additional
embodiments the complexes are administered at least 2 days, at
least 3 days, at least 4 days, at least 5 days, at least 6 days, at
least 7 days, at least 8 days, at least 9 days, at least 10 days,
at least 11 days, at least 12 days, at least 13 days or at least 14
days prior to the potential exposure to the organophosphate agent.
Suitably, the complex is administered at least once a week, more
suitably at least twice a week, etc., prior to potential exposure
to the OP agent. In further embodiments, the administration can be
once every 10-14 days, prior to potential exposure to the OP
agent.
[0111] In further embodiments, methods of treating toxicity
associated with exposure to an organophosphate agent in a human are
provided. Such methods suitably comprise administering intranasally
or via aerosol inhalation to the human a cationic liposome complex
as described herein. Suitably the cationic liposome complex
comprises a cationic liposome, an anti-transferrin receptor single
chain Fv (TfRscFv) directly complexed with, but not chemically
conjugated to, the cationic liposome, a nucleic acid molecule
encoding butyrylcholinesterase (BChE) contained in a first plasmid
associated with the cationic liposome, and a nucleic acid molecule
encoding a polyproline rich peptide contained in a second plasmid
associated with the cationic liposome. Suitably the TfRscFv and the
cationic liposome are present at a ratio in the range of about 1:20
to about 1:40 (w:w) and the nucleic molecules are present at a
ratio of about 1:5 to about 1:20 (.mu.g nucleic acid:.mu.g
liposome). In embodiments the complex is administered so as to
treat toxicity associated with exposure of at least
1.times.LD.sub.50 of the organophosphate agent.
[0112] Also provided are methods of preventing toxicity associated
with exposure to an organophosphate agent in a human. Such methods
suitably comprise administering intranasally or via aerosol
inhalation to the human a cationic liposome complex. Suitably the
cationic liposome complex comprises a cationic liposome, an
anti-transferrin receptor single chain Fv (TfRscFv) directly
complexed with, but not chemically conjugated to, the cationic
liposome, a nucleic acid molecule encoding butyrylcholinesterase
(BChE) contained in a first plasmid associated with the cationic
liposome, and a nucleic acid molecule encoding a polyproline rich
peptide contained in a second plasmid associated with the cationic
liposome. Suitably the TfRscFv and the cationic liposome are
present at a ratio in the range of about 1:20 to about 1:40 (w:w)
and the nucleic molecules are present at a ratio of about 1:5 to
about 1:20 (.mu.g total nucleic acid:.mu.g liposome). Suitably the
complex is administered so as to prevent toxicity associated with
exposure of at least 1.times.LD.sub.50 of the organophosphate
agent.
[0113] As described throughout, suitably the nucleic acid molecule
encoding BChE is contained in a first plasmid construct,
comprising, from 5' to 3': (a) at least one human adenovirus
enhancer sequence; (b) a cytomegalovirus (CMV) promoter; (c) a
multiple cloning site; (d) the nucleic acid molecule encoding BChE;
and (e) an SV 40 poly A sequence, wherein the 3' end of the plasmid
construct does not comprise adenovirus map units 9-16 when compared
to a wild-type adenovirus. In embodiments the nucleic acid molecule
encoding the polyproline rich peptide is contained in a first
plasmid construct, comprising, from 5' to 3': (a) at least one
human adenovirus enhancer sequence; (b) a cytomegalovirus (CMV)
promoter; (c) a multiple cloning site; (d) the nucleic acid
molecule encoding the polyproline rich peptide; and (e) an SV 40
poly A sequence, wherein the 3' end of the plasmid construct does
not comprise adenovirus map units 9-16 when compared to a wild-type
adenovirus.
[0114] In embodiments, BChE is a mutant version of BChE, such as a
G117H mutant.
[0115] Exemplary lipids, ratios of nucleic acids to liposomes and
exemplary ratios of the nucleic acids associated with the liposomes
are described herein.
[0116] Suitably the nucleic acid molecules are present at a molar
ratio of about 10:1 to about 1:10 (moles nucleic acid molecule
encoding butyrylcholinesterase (BChE):moles nucleic acid molecule
encoding a polyproline rich peptide), more suitably the nucleic
acid molecules are present at a molar ratio of about 5:1 to about
1:5 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide), most suitably the nucleic acid molecules are present at a
molar ratio of about 4:1 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide) or about 2 mole of one or more nucleic
acid molecules encoding butyrylcholinesterase (BChE), to about 1
mole of one or more nucleic acid molecules encoding a polyproline
rich peptide, or about 1 mole of one or more nucleic acid molecules
encoding butyrylcholinesterase (BChE), to about 1 mole of one or
more nucleic acid molecules encoding a polyproline rich
peptide.
[0117] Suitably the nucleic acid molecules are encapsulated within
the interior of the cationic liposome.
[0118] As described herein, suitable the complex is administered so
as to treat toxicity associated with exposure of up to
5.times.LD.sub.50 of the organophosphate agent. In embodiments, the
complex is administered immediately after exposure to the
organophosphate agent.
[0119] As described herein, suitably the complex is administered so
as to prevent toxicity associated with exposure of up to
5.times.LD.sub.50 of the organophosphate agent. Suitably the
complex is administered at least 6 hours prior to potential
exposure to the organophosphate agent. In embodiments, the complex
is administered at least once a week prior to potential exposure to
the organophosphate agent.
[0120] Also provided herein are methods of delivering
butyrylcholinesterase (BChE) to the bloodstream of a mammal (e.g.,
a human) comprising administering intranasally or via aerosol
inhalation to the mammal a cationic liposome complex. As described
herein, suitably the cationic liposome complex comprises a cationic
liposome, an anti-transferrin receptor single chain Fv (TfRscFv)
directly complexed with, but not chemically conjugated to, the
cationic liposome, a nucleic acid molecule encoding
butyrylcholinesterase (BChE) contained in a first plasmid
associated with the cationic liposome, and a nucleic acid molecule
encoding a polyproline rich peptide contained in a second plasmid
associated with the same or different cationic liposome.
[0121] Suitably the TfRscFv and the cationic liposome are present
at a ratio in the range of about 1:20 to about 1:40 (w:w) and the
nucleic molecules are present at a ratio of about 1:5 to about 1:20
(.mu.g nucleic acid:.mu.g liposome).
[0122] In embodiments of the various methods described herein,
suitably the complex is administered so as to result in an amount
of BChE in the bloodstream of the human of at least 250 mg/70 kg
(weight of BChE/weight of the human). Suitably, the complex is
administered so as to result in an amount of BChE in the
bloodstream of the human of at least 200 mg/70 kg (weight of
BChE/weight of the human), more suitably at least 225 mg/70 kg, at
least 250 mg/70 kg, at least 275 mg/70 kg, at least 300 mg/70 kg,
at least 325 mg/70 kg at least 350 mg/70 kg, or at least 400 mg/70
kg.
[0123] As described throughout, suitably the nucleic acid molecule
encoding BChE is contained in a first plasmid construct,
comprising, from 5' to 3': (a) at least one human adenovirus
enhancer sequence; (b) a cytomegalovirus (CMV) promoter; (c) a
multiple cloning site; (d) the nucleic acid molecule encoding BChE;
and (e) an SV 40 poly A sequence, wherein the 3' end of the plasmid
construct does not comprise adenovirus map units 9-16 when compared
to a wild-type adenovirus. Suitably the nucleic acid molecule
encoding the polyproline rich peptide is contained in a first
plasmid construct, comprising, from 5' to 3': (a) at least one
human adenovirus enhancer sequence; (b) a cytomegalovirus (CMV)
promoter; (c) a multiple cloning site; (d) the nucleic acid
molecule encoding the polyproline rich peptide; and (e) an SV 40
poly A sequence, wherein the 3' end of the plasmid construct does
not comprise adenovirus map units 9-16 when compared to a wild-type
adenovirus.
[0124] Alternatively the nucleic acid molecule encoding BChE, and
the nucleic acid molecule encoding the polyproline rich peptide,
are in the same construct, but the nucleic acid molecule encoding
BChE is placed downstream from the high expression promoter
disclosed in U.S. Published Patent Application No. 2007/0065432,
while the nucleic acid molecule encoding the polyproline rich
peptide is placed downstream of a standard promoter such as RSV or
CMV
[0125] In embodiments, the BChE is a mutant version of BChE, such
as a G117H mutant.
[0126] Exemplary compositions of the liposomes and ratios for the
various components are described herein. Suitably, the nucleic acid
molecules are present at a molar ratio of about 10:1 to about 1:10
(moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide).
[0127] Suitably the nucleic acid molecules are present at a molar
ratio of about 10:1 to about 1:10 (moles nucleic acid molecule
encoding butyrylcholinesterase (BChE):moles nucleic acid molecule
encoding a polyproline rich peptide), more suitably the nucleic
acid molecules are present at a molar ratio of about 5:1 to about
1:5 (moles nucleic acid molecule encoding butyrylcholinesterase
(BChE):moles nucleic acid molecule encoding a polyproline rich
peptide), most suitably the nucleic acid molecules are present at a
molar ratio of about 4:1 (moles nucleic acid molecule encoding
butyrylcholinesterase (BChE):moles nucleic acid molecule encoding a
polyproline rich peptide) or about 2 mole of one or more nucleic
acid molecules encoding butyrylcholinesterase (BChE), to about 1
mole of one or more nucleic acid molecules encoding a polyproline
rich peptide, or about 1 mole of one or more nucleic acid molecules
encoding butyrylcholinesterase (BChE), to about 1 mole of one or
more nucleic acid molecules encoding a polyproline rich
peptide.
[0128] Suitably the liposome complexes described herein comprise
two co-encapsulated plasmid vectors carrying the BChE genes (e.g.,
mutant BChE) and polyproline rich peptide genes, produce a level of
tetrametic BChE (at least 40%, more preferably 70%-90% tetramer
form) that is active in circulation for 1 to 21 days. The methods
described herein suitably produce enzyme levels that obtain a
therapeutically effective amount. It is unexpected that the complex
of this invention encapsulating a plasmid vector encoding for BChE
and a plasmid vector encoding for a polyproline rich peptide under
the control of a high expression promoter can achieve the level of
activity, and length of protein expression of active BChE.
[0129] It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein may be made without
departing from the scope of the invention or any embodiment
thereof. Having now described the present invention in detail, the
same will be more clearly understood by reference to the following
examples, which are included herewith for purposes of illustration
only and are not intended to be limiting of the invention.
Example 1
Presence of Transgene (Exogenous p53) in Metastatic Tumors from
Subjects in a Phase I Clinical Trial of scL Delivered Wtp53
Gene
[0130] This example demonstrates a successful transgene delivered
by the scL nanocomplex (a liposome composition of the present
invention comprising total nucleic acid, lipid and the single chain
antibody TfscFv at a weight ratio of about 1:10:0.33 (.mu.g total
nucleic acid:.mu.g lipid:.mu.g single chain antibody) (also
referred to herein as scL), encoding exogenous wtp53 DNA, SGT-53,
is present in the tumors of treated patients. To assess tumor
delivery of SGT-53, DNA PCR was performed to determine the
exogenous p53 gene delivered by the SGT-53 complex in the tumors
from subjects in an open label, single center, sequential dose
escalating, Phase 1 study evaluating the safety, pharmacokinetics,
and potential activity of SGT-53 in subjects with solid tumors and
who had been offered all standard or approved therapies. The doses
of SGT-53 administered to the subjects escalated from 0.6 mg
DNA/infusion to 3.6 mg DNA/infusion. Study drug was administered
twice weekly for five weeks for a total of ten infusions. In 2
subjects a biopsy of the most accessible tumor was performed within
24 to 96 hours after the last administration of SGT-53. At least 20
mg of tumor tissue was obtained. A microscopic examination was
performed on the frozen sample to ensure that the sample contained
tumor. The tumor tissue was snap frozen and was used for DNA PCR to
identify the presence and level of exogenous wtp53 DNA in the
tumor. In both cases, the tumor sample was obtained from a
metastatic lesion. A 700 bp fragment was amplified using one primer
in the vector and one primer in the p53 insert. Using this
approach, only the exogenous p53 transgene was amplified. Tumor
biopsies were obtained from patients after the end of treatment
with the lowest dose (0.6 mg/infusion) (T1) and with the 3.6
mg/infusion dose (T2). As shown in FIG. 1, the amplified 700 bp
fragment of the exogenous p53 gene was clearly present in both
tumors. More importantly, the transgene was present in a dose
dependent manner as would be expected with targeted delivery.
Example 2
Increase in Protein Expression after Cloning into the pSCMV
Vector
[0131] The increase in protein expression as a result of placing a
gene under the control a high expression promoter, as disclosed
herein, is shown in FIG. 2. Human cells were transfected with scL
(prepared as described below in Example 7) carrying a gene cloned
into the pSCMV vector, or in the original construct. Twenty-four
hours post-transfection, expression of the specific gene was
assessed by Western analysis. The purified protein expressed from
this specific gene is included on the gel as a positive control and
for verification of protein positioning. An at least 10 fold higher
level of the specific protein expression was observed with the
pSCMV clone (X455) compared to that of the original construct
(X457) which uses a standard promoter (FIG. 2). The specific band
is present in X457 upon longer exposure (data not shown). GAPDH
levels demonstrate equal protein loading. UT=untreated cells;
+Con=purified protein to confirm position.
Example 3
Ability of the scL Liposome Complex to Cross the Blood-Brain
Barrier and Target Neuronal Cells
[0132] The ability to cross the blood-brain barrier and target
neuronal cells in the brain is shown in FIGS. 3 and 4. In FIG. 3,
Balb/C mice were injected with scL carrying either the pSCMV high
expression plasmid containing the GFP gene (FIG. 3A) or carrying a
fluorescently labeled oligonucleotide (6-FAM-ODN) (FIG. 3B)
prepared as described below in Example 7. 24 or 48 hours later the
brains were excised and imaged using the Maestro.RTM. In Vivo
Imaging System (Perkin Elmer). In both cases, a significantly
higher level of accumulation of the fluorescence signal is observed
in the brains of the mice injected with the scL-delivered payload
when compared to the level of accumulation/signal with either Free
(unencapsulated) GFP DNA or Free (unencapsulated) 6-FAM-ODN.
[0133] The ligand-liposome complex targeting the TfR and carrying a
plasmid encoding GFP (100 .mu.g cDNA) prepared as described below
in Example 7 was injected via a catheterized jugular vein in rats.
After 24-36 hours of post-surgery recovery, animals were sacrificed
and coronal 40 .mu.m brain sections analyzed via fluorescence
microscopy. As expected, non-targeted liposome-GFP produced no
detectable EGFP(+) cells in the brain (FIG. 4 B). In contrast,
Tf-Lip-DNA injections yielded widespread EGFP expression (mainly in
neuronal cells) in cortex and hippocampus, which indicate that
TfR-targeted vectors cross the blood-brain barrier to deliver the
genetic payload to adult neurons in vivo. Numerous EGFP(+) neurons
(arrow) and neuropil (arrow) are observed in hippocampus (CA1 area)
(A1) and cortex (A2) following iv injection of Tf-Lip-EGFP cDNA.
Parvalbumin IF staining revealed innervation of EGFP(+)
neurons.
Example 4
Enhanced Luciferase Expression and Tissue Uptake by scL Delivery
after Intranasal Administration
[0134] The Luciferase gene, either as Free (unencapsulated), or
scL-encapsulated plasmid DNA, prepared as described below in
Example 7, was intranasally administered to Balb/c mice. 24 hours
post intranasal administration the mice were intraperitoneally
injected with Luciferin and imaged with a IVIS.RTM. (Xenogen) In
Vivo Imaging system (Perkin Elmer). The difference in the level of
gene expression is shown in FIG. 5.
[0135] In these mouse experiments the timing and level of uptake of
an scL delivered reporter after intranasal administration were
assessed. The scL nanocomplex prepared using the ratios disclosed
above encapsulating an FITC labeled molecule (100 .mu.g) was
intranasally administered to immune competent mice. At times from
6-48 hrs post-administration, animals were humanely euthanized.
Liver, lung and brain tissues were excised and subjected to Flow
cytometry to determine the number of FITC positive cells in each
organ at each time point. For all three organs the percent of FITC
positive cells was found to peak between 16 and 30 hours. The % of
cells in each tissue that were transfected at the peak time varied.
Not unexpectedly, the largest % of FITC positive cells was found in
the lungs (.about.20-40%) while 3-4% positive cells were found in
the brain and .about.1.5% of the liver cells carried the scL
delivered molecule. However, it should be noted that, since the
liver is a large organ, based upon Marino (20), 1.5% of liver cells
is .about.2.25.times.10.sup.7 cells, a relatively large number.
Similarly, 20-40% positive cells in the lung represents
.about.5.7.times.10.sup.7 cells, while 3-4% positive cells in brain
equals .about.4.4.times.10.sup.6 cells.
Example 5
In Vitro Induction of ERKI/II Expression by Carbachol
[0136] Carbachol is a drug that binds and activates the
acetylcholine receptor (agonist). Thus treatment with Carbachol
mimics the effects of OP agents. Carbachol has been shown to induce
expression of ERKI/II in brain (21). That treatment of lung cancer
cells with Carbachol can induce expression of ERKI/II and that
pre-treatment with atropine, a competitive inhibitor (antagonist)
of the muscarinic acetylcholine receptors can inhibit this
induction (FIG. 6). Human A549 cells were used as a model to test
these responses. A549, with or without 30 min pre-treatment with
100 uM atropine (final concentration), were treated with 100 .mu.M
(final concentration) Carbachol. Thirty minutes later the cells
were harvested, protein isolated and 60 .mu.g total cellular
protein analyzed by Western using an antibody that detects both
ERKI and ERKII proteins. Lane 1=untreated (uninduced) cells showing
low basal levels of the proteins; Lane 3=Induced expression after
Carbachol treatment; Lane 2=Lack of ERKI/II induction after
pre-treatment with Atropine followed by Carbachol treatment. Thus,
inhibition of this Carbachol induced expression of ERKI/II can be
used as a downstream molecular surrogate to assess the
effectiveness of scL-mtBChE/scL-ppol treatment both in vitro and in
vivo.
Example 6
Cloning into High Expression Vector pSCMV
[0137] Plasmid vectors containing either the wt human BChE
(huBChE), the G117H mutant version of human BChE (mtBChE) (see,
e.g., McTiernan, et al., "Brain cDNA clone for human
cholinesterase," Proc. Natl. Acad. Sci. 84:6682-6686, FIG. 2, the
disclosure of which is incorporated by reference herein in its
entirety for all purposes) or a polyproline rich peptide (ppro)
(see Altamirano and Lockridge, Chemico-Biological Interactions
119-120:53-60 (1999) (see page 57, section 2.9); Krejci et al., The
Journal of Biological Chemistry 272:22840-22847 (1997) (see page
22842, FIG. 1); and Bon et al., The Journal of Biological Chemistry
272:3016-3021 (1997) (see section bridging pages 3016-3017) the
disclosures of which are incorporated by reference herein in their
entireties) downstream of the high expression promoter disclosed in
U.S. Published Patent Application No. 2007/0065432 have been
prepared. The plasmid vector (pSCMV) results in levels of
expression of the gene of interest 2-10 fold higher than with
standard constructs. This high expression plasmid, pSCMV, is
derived from the pBR322 plasmid backbone and also contains a
multiple cloning site, and a gene conferring resistance to the
antibiotic Kanamycin. This plasmid has been prepared to carry two
different tumor suppressor genes under cGMP condition for use in
human clinical trials. Thus, the construct is acceptable to the FDA
for use in humans. Using standard procedures, and restriction sites
well known by those familiar with the art, the 1.8 Kb BChE and
.about.300 Bp ppro inserts (11) from the original vectors were
subcloned into the multiple cloning site in pSCMV (FIG. 7). The
resulting plasmids were propagated in commercially available
bacterial host TOP 10 F.sup.1 (Invitrogen, Carlsbad, Calif.).
Plasmid DNA was then prepared using standard methods well known in
the art. The resultant plasmids were tested. Analysis indicated a
260:280 ratio of >1.9, the presence of >75% supercoiled
molecules in the population (FIG. 8), and undetectable levels of
endotoxin.
Example 7
In Vitro Characterization of the Clones Produced in Example 6
[0138] The pSCMV plasmids carrying either the huBChE, mtBChE or
ppro genes are characterized for level of BChE activity, T.sub.1/2
and to determine the optimum ratio of BChE to ppro for activity and
tetramerization. For the studies described below a commercially
available (from ATCC) cell line, CHO (Chinese hamster ovary), human
lung cancer A549 or human prostate cancer DU145 cells are employed.
CHO, which has a basal level of BChE activity, is used for
continuity purposes since these cells were previously employed by
other investigators, including Lockridge and collaborators, in the
study of BChE activity (11).
[0139] Assessing Level of BChE Expression
[0140] The pSCMV-wtBChE or the pSCMV-mtBChE vectors are
encapsulated in the scL nanocomplex together with the pSCMV-ppro
vector to form scL-wtBChE/ppro and scL-mtBChE/ppro complexes. The
vectors are mixed for encapsulation at a 4:1 molar ratio
(pSCMV-mtBChE to pSCMV-ppro) of the inserts. The ratios of TfRscFv
ligand, liposome and total plasmid DNA are 0.33 ug to 10 ug to 1 ug
(TfRscFv to liposome to DNA). The complex is formed by simple
mixing of components at a defined ratio (32).
TABLE-US-00002 Exemplary liposome components LipA DOTAP/DOPE 1:1
molar ratio LipB DDAB/DOPE 1:1 molar ratio LipC DDAB/DOPE 1:2 molar
ratio LipD DOTAP/Chol 1:1 molar ratio LipE DDAB/Chol 1:1 molar
ratio LipG DOTAP/DOPE/Chol 2:1:1 molar ratio LipH DDAB/DOPE/Chol
2:1:1 molar ratio (DOTAP = dioleoyltrimethylaminnonium phosphate,
DDAB = dimethyldioctadecylammonium bromide; DOPE =
dioleoylphosphatidylethanolamine; chol = cholesterol).
[0141] Liposomes are prepared by the ethanol injection method
modified from that described by Campbell, M J (Biotechniques 1995
Jun. 18(6):1027-32). In brief, all lipids are solubilized in
ethanol and mixed, injected into vortexing pure water of
50-60.degree. C. with a Hamilton syringe. The solution is vortexed
for a further 10-15 min. The final concentration is 1-8 mM total
lipids.
[0142] The TfRscFv-immunoliposome complexes (other ligands can also
be used as described herein) are prepared by mixing the TfRscFv
with liposome composition A (or any of the liposome compositions
given above) at defined ratios of single chain protein to liposome
and DNA in the mixed complex. The preparation of the complexes is
in accordance with the following general procedure.
[0143] The appropriate amount of 2 mM to 8 mM liposome (A-H
described above) is mixed with any water (e.g., DI water) required
to give a desired volume and gently inverted 10 times to mix, or
for larger volumes rotated at 20-30 RPM for 1-2 minutes. To the
liposome-water mixture, the appropriate amount of TfRscFv, antibody
or antibody fragment is added to give the desired ratio and mixed
by gentle inversion for about 5-10 seconds or for larger volumes
rotated at 20-30 RPM for 1-2 minutes. This mixture is kept at room
temperature for 10-20 minutes (again inverted gently for 5-10
seconds after approximately 5 minutes). At the same time, the
appropriate amount of DNA comprising the, the pSCMV-wtBChE vector,
or pSCMV-mtBChE vector and pSCMV-ppro vector constructs (at a molar
ratio of the BChE to ppro inserts of 10:1 to 1:10, more preferably
5:1 to 1:5, most preferably 4:1, 2:1 or 1:1) is mixed by inversion
for 5-10 seconds, or for larger volumes rotated at 20-30 RPM for
1-2 minutes, with any water required to give a desired volume.
Typically, for in vivo use, it is desirable to provide about 5
.mu.g to about 100 mg of DNA per administration (includes IN, oral,
Aerosol, IV, IP, IM etc).
[0144] The DNA solution is quickly added to the TfRscFv (or
antibody or antibody fragment)-liposome solution and the mixture is
inverted for 5-10 seconds or for larger volumes rotated at 20-30
RPM for 1-2 minutes. The final mixture is kept at room temperature
for 10-20 minutes, gently inverting again for 5-10 seconds after
approximately 5 minutes. For use in vivo, 50% dextrose or 50%
sucrose is added to a final concentration of 5-20% (V:V) and mixed
by gentle inversion for 5-10 seconds, or for larger volumes rotated
at 20-30 RPM for 1-2 minutes. A specific example at a suitable
ratio (w:w) of TfRscFv to Liposome to DNA of 0.33 .mu.g to 10 .mu.g
to 1 .mu.g is as follows. For 40 .mu.g of total DNA in a final
volume of 800 mix 183 .mu.l water with 280 .mu.l of 2 mM liposome
solution. Add 34 .mu.l of TfRscFv (with a concentration of 0.4
.mu.g/ml). Mix 183 .mu.l water with 40 .mu.l of 1 .mu.g/1 .mu.l
DNA. Add 80 .mu.l of 50% Dextrose or 160 .mu.l of 50% Sucrose as
the last step.
[0145] When the {K[K(H)KKK]5-K(H)KKC} peptide (HoKC) (SEQ ID NO: 1)
is included in the complex, the cationic liposomal formulations A
(DOTAP:DOPE at a 1:1 molar ratio), B (DDAB:DOPE at 1:1), G
(DOTAP:DOPE:cholesterol at 1:1:1) and H (DDAB:DOPE:cholesterol at
1:1:1) (or any of the liposomes formulations given above) are
prepared using the ethanol injection method as described above.
Each liposome formulation also includes MPB-DOPE at 5 molar percent
of total lipid. Since the HoKC peptide carries a terminal cysteine,
MPB-DOPE is included in all of the liposome compositions to allow
conjugation of peptide to the liposome. The Lip-HoKC liposomes are
prepared using a coupling reaction between the cationic liposomes
carrying the maleimide group (Lip-MPB) and the peptide as follows.
An aliquot of 0.1 mmol of the peptide with a free thiol group on
cysteine is added to 2 mmol of Lip-MPB in 10 mM HEPES, pH 7.4,
solution and rotated at room temperature (20-30 r.p.m.) for 2 h.
The resulting Lip-HoKC has a lipid concentration of 1.4 mM.
[0146] When the liposome comprises HoKC, the full complex is formed
in a manner identical to that used to produce the TfRscFv-Lip-DNA
complex without HoKC. Here also the TfRscFv or any antibody or
antibody fragment (including Fab' or Mab) is mixed with Lip-HoKC
(by gently inversion 10 times or for larger volumes rotated at
20-30 RPM for 1-2 minutes) at a specific ratio, and incubated at
room temperature for 10-20 min. DNA is then added to the TfRscFv
(or antibody or antibody fragment)-Lip-HoKC solution, mixed by
gently inversion 10 times or for larger volumes rotated at 20-30
RPM for 1-2 minutes, and again incubated at room temperature for
10-20 min, after which dextrose or sucrose is added to a final
concentration of 5-20%, mixed by gentle inversion 10 times, or for
larger volumes rotated at 20-30 RPM for 1 minute, and incubated at
room temperature for 10-20 minutes. The ratio of the
TfRscFv:LipA-HoKC:DNA in the complex is 0.3 mg:7 nmol:1 mg.
[0147] Dynamic Laser Light Scattering (DLS) with a Malvern
Zetasizer NanoZS is employed to measure the size of the scL
nanocomplexes. The size of the complex suitably is less than 400
nm. This instrument also has the capability to measure
zeta-potential, a determinant of the overall charge of the
nanocomplex. For these complexes the charge is suitably positive,
between about 20 and 50 mV. Modifying the ratios of the components
to control the size, charge and/or activity of the scL-wtBChE/ppro
or scL-mtBChE/ppro can readily be carried out as desired.
[0148] The sizes of the nanocomplexes prepared as described above
are shown in Table 2
TABLE-US-00003 Molar Ratio Size (BChE to ppro) scL-pSCMV wtBChE
114.7 nm -- scL-pSCMV mtBChE 134.5 nm -- scL-CMV mtBChE 105.3 nm --
scL-pSCMV mtBChE/ppro 105.1 nm 2:1 96.4 nm 4:1 scL-CMV mtBChE/ppro
97.1 nm 2:1 72.1 nm 4:1 The Zeta potentials were in the range of
20-40.
[0149] CHO cells, DU145 or A549 cells are plated at
.about.2.times.10.sup.5 cells/well of a 6 well plate or
2.times.10.sup.4 cells/well of a 24 well plate. The total DNA dose
for transfection in these initial experiments is 0.1 to 2
.mu.g/well. This dose is based upon prior transfection experience
which has shown that this dose is often the most appropriate when
transfecting this number of cells. 24 hrs later the cells are
transfected with the scL-wtBChE/ppro or the scL-mtBChE/ppro
complexes prepared using the method as described above. The CHO
cells are also transfected with scL encapsulating the original
vector constructs which have the unmodified CMV or RSV promoters to
serve as controls. Untransfected cells are also included in all
experiments. Based upon previous experience with the pSCMV plasmid,
there is significant protein expression within 24 hours. Thus,
.about.24 hours post-transfection the relative amounts of BChE
tetramers, dimers and monomers in each of the samples (in both the
cells and the supernatant) are assessed by non-denaturing
polyacrylamide gel electrophoresis on 4-30% gradient gels and
staining with 2 mM butyrylthiocholine by the method of Karnovsky
and Roots (23, 33). Purified plasma huBChE is included as a
control. At least a 2 fold increase in BChE protein detected after
transfection is considered a positive result.
[0150] Examples of the results of such experiments are shown in
FIGS. 9-11. CHO cells or A549 cells were plated at
.about.2.times.10.sup.4 cells/well of a 24 well plate. The scL
nanocomplex was prepared encapsulating a plasmid encoding the
mutant BChE plus a second plasmid encoding the ppro gene. In these
plasmids the gene was under the control of a standard
cytomegalovirus (CMV) promoter or under the control of the high
expression vector (pSCMV) described in this application.
[0151] In the experiments with the CHO-K1 cells, the complexes were
prepared using the method as described above. The total DNA in each
scL nanocomplex was 1 ug/well in 30 ul For the scL nanocomplex
encapsulating the vector carrying the mtBChE gene under the control
of the pSCMV promoter and the and the vector carrying the ppro gene
under the control of the pSCMV promoter, 22.4 .mu.l of 2 mM
liposome solution was added to 63.5 ul water. The liposome-water
was mixed with 5.08 .mu.l of TfRscFv (with a concentration of 0.34
.mu.g/ml). To the cationic liposome-TfRscFv mixture was added 3.2
.mu.g of total DNA (the specific BChE plus ppro vectors) in a final
volume of 5.02 .mu.l. Of the final scL nanocomplexes 3, 9 or 18
.mu.l (representing 0.1, 0.3 and 0.6 ug total DNA, respectively) of
the scL nanocomplexes were added to the cells.
[0152] In these experiments the molar ratio of the BChE plasmid DNA
to the ppro plasmid DNA was 4:1. The expression of exogenous BChE
was assessed by non-denaturing polyacrylamide gel electrophoresis
on 4-30% gradient gels and staining with 2 mM butyrylthiocholine by
the method of Karnovsky and Roots (23, 33). As shown in FIG. 9 with
CHO-K1 cells transfected with scL-mtBChE/ppro nanocomplex, there is
a DNA dose dependent increase in expression of the BChE evident.
Unexpectedly, nearly 100% of the BChE was present in the active
Tetramer form.
[0153] A549 cells were transfected with either scL-CMV mtBChE/ppro
or scL-pSCMV mtBChE/ppro prepared using the method as described
above. For the scL nanocomplex encapsulating the vector carrying
the mtBChE gene under the control of the CMV promoter and the and
the vector carrying the ppro gene under the control of the CMV
promoter, or the scL nanocomplex encapsulating the vector carrying
the mtBChE gene under the control of the pSCMV promoter and the and
the vector carrying the ppro gene under the control of the pSCMV
promoter 14 .mu.l of 2 mM liposome solution was added to 32.83 ul
water. The liposome-water was mixed with 3.17 .mu.l of TfRscFv
(with a concentration of 0.21 .mu.g/ml). To the cationic
liposome-TfRscFv mixture was added 3.2 .mu.g of total DNA (the
specific BChE plus ppro vectors) in a final volume of 10.19 (CMV
promoter) or 31.39 .mu.l (pSCMV promoter). Enough Serum Free Media
was added to bring the final volume to 100 ul. Of the final scL
nanocomplexes 25 ul (representing 0.5 ug total DNA) of the scL
nanocomplexes was added to the cells. In these experiments the
molar ratio of the BChE plasmid DNA to the ppro plasmid DNA was
4:1.
[0154] The level of BChE expression with each of the scL complexed
plasmids was assessed at two time points by non-denaturing
polyacrylamide gel electrophoresis on 4-30% gradient gels and
staining with 2 mM butyrylthiocholine by the method of Karnovsky
and Roots (23, 33). As shown in FIG. 10 (16 days post-transfection)
and FIG. 11 (28 days post-transfection), at both time points there
is a significantly higher level of BChE expression in the cells
transfected by the scL carrying the BChE and ppro under control of
the high expression pSCMV promoter compared to those transfected
with the plasmids under control by standard CMV promoter.
Unexpectedly, this increase in expression is even greater as time
goes on (28 days).
[0155] Assessing Level of BChE Activity
[0156] Transfection of CHO DU145 and A549 cells with
scL-wtBChE/ppro, or scL-mtBChE/ppro, with the plasmids under the
control of either the high expression pSCMV promoter or the
standard CMV promoter, and with the controls described above, are
performed. 24 hours post-transfection, the level of BChE activity
in the cells and supernatant is determined by means of the Ellman
assay (34,35). BChE activity is determined with 1 mM
butyrylthiocholine in 0.1M potassium phosphate, 0.5 mM
dithiobisnitrobenzoic acid at 25.degree. C., monitoring absorbance
at 412 nm. Functional activity is calculated using the molar
extinction coefficient of 13,600 M.sup.-1 cm.sup.-1. These
experiments are designed to confirm the increased activity of mt
over wt BChE clones while in the pSCMV vectors, analogous to the
previously reported findings with the original unmodified vectors
(10,11). At least a 2 fold increase in BChE activity with the
pSCMV-mtBChE clone as compared to the pSCMV-wt BChE clone is
desirable.
[0157] Examples of such experiments in DU145 cells are shown below.
Different molar ratios of the pSCMV plasmid DNAs encoding mtBChE to
the pSCMV plasmid DNAs encoding ppro in the scL nanaocomplex were
performed in DU145 cells. In these experiments molar ratios of 2:1
(Table 3) and 4:1 (Table 4) were used. 2.5.times.10.sup.4 DU145
cells were seed/well of a 24 well plate and were transfected 24
hours later. The scL nanocomplexes were prepared using the same
ratio of 0.33 ug TfRscFv to 10 ug Liposome to 1 ug total DNA using
the procedure described above in Example 7. For each of the scL
nanocomplexes, 2 .mu.g of total DNA (pSCMV-BChE and pSCMV-ppro at
the appropriate ratios) in a final volume of 5.3 .mu.l (with
additional 1.times.Tris:EDTA buffer as needed to make 5.3 ul) was
used. To 3.17 .mu.l of TfRscFv (with a concentration of 0.34
.mu.g/ml) was added to 4.7 ul water and 14 .mu.l of 2 mM liposome.
To this TfRscFv-Liposome mixture was added the 5.3 ul of DNA from
above. 3 ul of Serum Free Medium was added and the resulting mixed
by gentle inversion prior to addition to the cells. The total DNA
in each scL nanocomplex was 0.5 ug/well in 25 ul. The levels of
BChE activity assessed by Ellman assay is shown below in Tables 3
and 4.
TABLE-US-00004 TABLE 3 Transfection of DU145 Cells 0.5 ug DNA
(mtBChE:ppro Ratio = 2:1): Days ACTIVITY Post-Transfection
CONSTRUCT (U/mL .times. 10.sup.-3) 5 scL-CMV 1.51 scL-pSCMV 2.15 7
scL-CMV 2.96 scL-pSCMV 3.68 9 scL-CMV 4.49 scL-pSCMV 5.38
TABLE-US-00005 TABLE 4 Transfection of DU145 Cells 0.5 ug DNA
(mtBChE:ppro Ratio = 4:1): Days ACTIVITY Post-Transfection
CONSTRUCT (U/mL .times. 10.sup.-3) 13 scL-CMV 1.367 scL-pSCMV 1.416
18 scL-CMV 1.175 scL-pSCMV 1.342
[0158] At both ratios, the level of BChE activity is higher after
transfection when the genes are cloned into the high expression
promoter then when they are under the control of the standard CMV
promoter showing the difference between this approach and that
currently in use by others in the field.
[0159] After confirming that the mtBChE clone has higher activity
than wt, only the mtBChE clone is employed in additional
experiments. After confirming that the genes cloned into the
constructs under the control of the high expression promoter have
higher activity than when under the control of the standard CMV
promoter, only the these clones are employed in additional
experiments.
[0160] Determining Optimal Molar Ratio
[0161] The molar ratio of the pSCMV-mtBChE insert to pSCMV-ppro
insert in the scL complex for transfection of the CHO and/or A549
cells is varied to establish the ratio that results in the highest
amount of tetramers and activity. The molar ratios (pSCMV-mtBChE to
pSCMV-ppro) are 4:1; 1:1 and 1:4. 24 hours post-transfection, the
cells and supernatant are harvested and the activity determined via
the Ellman assay. The relative amount of tetramers, dimers and
monomers are assessed via non-denaturing gel electrophoresis as
described above. It is desirable that the scL-mtBChE/ppro inserts
yield at least 40%, more suitably 70%-90% of the tetramer form.
Modification of the ratios can be utilized to achieve a desired
level of tetramers.
[0162] Experiments using different molar ratios of the pSCMV
plasmid DNAs encoding mtBChE to the pSCMV plasmid DNAs encoding
ppro in the scL nanaocomplex were performed in DU145 cells. In
these experiments molar ratios of 2:1, 1:1 or 1:2 (BChE:ppro)
(Table 5) were used. 2.5.times.10.sup.4 DU145 cells were seed/well
of a 24 well plate and were transfected 24 hours later. The scL
nanocomplexes were prepared using the same ratio of 0.33 ug TfRscFv
to bug Liposome to 1 ug total DNA using the procedure described
above in Example 7. To 1 .mu.l of TfRscFv (with a concentration of
0.34 .mu.g/ml) was added 10 ul water. To this was added 7 .mu.l of
2 mM liposome solution. For each of the scL nanocomplexes, 1 ng of
total DNA (pSCMV-BChE and pSCMV-ppro at the appropriate ratios) in
a final volume of 5 .mu.l was mixed with 15 ul of water then added
to the TfRscFv-liposome solution. 62 ul of Serum Free Medium was
then added and the resulting mixed by gentle inversion prior to
addition to the cells. The total DNA in each scL nanocomplex was
0.3 ug/well in 30 ul.
TABLE-US-00006 TABLE 5 Transfection of DU145 Cells at Different
Ratios of pSCMV-BChE to pSCMV-ppro in the scL Nanocomplex: Days
RATIO ACTIVITY Post-Transfection (BChE/ppro) (U/mL .times.
10.sup.-3) 12 UT 6.41 2:1 6.49 1:1 7.35 1:2 7.03 19 UT 5.63 2:1
6.57 1:1 5.98 1:2 5.61
[0163] Determining Peak and Duration of Expression/Activity
[0164] Based upon previous experience with the pSCMV plasmid,
significant protein expression begins .about.24 hours
post-transfection and continues for up to 4-5 days. Thus, to
determine the time of peak BChE expression and activity, CHO and/or
A549 cells are transfected with scL-mtBChE/ppro prepared at the
desired molar ratio. Daily from days 0 (pre-treatment) to 14
post-transfection the cells and supernatant are harvested, and the
level of BChE activity determined via Ellman assay. A significant
increase in the relative amount of the tetrameric form of BChE and
an activity curve and T.sub.1/2 similar to that of normal hBChE is
desired. mtBChE activity from between 1 to 14 days or even longer
is desirable. The results of experiments described above in
Examples 7 (FIGS. 10 and 11 and Tables 3-5) demonstrate an increase
in expression of the tetrameric form of BChE and the activity of
BChE over time using the scL nanocomplex of this application.
Example 8
Assessing Efficacy In Vitro
[0165] Carbachol is a drug that binds and activates the
acetylcholine receptor (agonist). Thus, treatment with Carbachol
mimics the effects of OP agents. As the use of chemical warfare
agents is strictly regulated, Carbachol can be used as a nerve
agent model compound. Carbachol induced the expression of ERKI/II
in human lung cancer A549 cells, and this induction is reversed by
pre-treatment with the muscarinic receptor antagonist atropine
(FIG. 6). In this Example, the ability of transfected
scL-mtBChE/ppro to protect against OPs in vitro, using expression
of ERKI/II as the surrogate end-point, is investigated.
[0166] A549 cells are transfected with scL-mtBChE/ppro at the
desired ratio. At the peak expression/activity time, the cells are
treated with 100 .mu.M Carbachol. 30 min later the cells are
harvested and ERKI/II expression levels determined by Western
analysis. Untreated cells and those treated only with Carbachol, or
just scL-mtBChE/ppro serve as controls. The cells are harvested,
and protein isolated as previously described (13). 40 .mu.g of
total protein is separated by SDS PAGE electrophoresis, transferred
to a Nylon membrane and probed with an antibody that detects both
ERKI and ERKII proteins, and a signal is detected using the ECL
Western blotting detection system (GE Lifesciences)
[0167] Prophylactic treatment with the scL-mtBChE/ppro suitably
inhibits the Carbachol induced expression of ERKI/II. Various doses
of the scL nanocomplexes are tested to achieve maximum inhibition
which is at least 50%, more preferentially 70%, most preferentially
90% or greater. If at least 50% inhibition is not observed, the
dose of the scL is suitably increased (no change in ratio) until it
is achieved. Moreover, at the time of Carbachol treatment, a
portion of the cells and supernatant are used to determine the
level of BChE activity by the Ellman assay to correlate activity
with down-modulation of ERKI/II.
Example 9
Establishing Baseline BChE Levels in Mice
[0168] A transgenic homozygous BChE knockout mouse (BChE-/-) as a
model for human BChE deficiency has been developed (29) and is
commercially available from Jackson Laboratories. These mice are in
a C57BL/6 background (BChE+/+). Thus, using published procedures
well known in the art (29), the baseline level of BChE activity in
plasma, lung, brain and liver tissues is determined by the Ellman
assay in the BChE-/- knockout and in C57BL/6 mice. The baseline
activity of BChE in the plasma, lung, brain and liver in the BChE
knockout mouse (BChE-/-) is suitably in the range of 0+0.005,
0.01+0.02, 0.02+0.02 and 0.09+0.05 U/ml (plasma and U/mg (tissues),
respectively. The baseline activity of BChE in the plasma, lung,
brain and liver in the C57BL/6 mice (BChE+/+) is suitably in the
range of 1.5+0.3, 0.38+0.2, 0.2+0.1 and 3.0+0.5 U/ml (plasma and
U/mg (tissues), respectively.
[0169] The levels of BChE activity in the plasma of normal,
untreated C57BL/6 mice (BChE+/+) was assessed by the Ellman Assay
as described above using well established protocols know in the
literature (34, 35). Two individual mice were tested. The
background level of BChE was found to be 0.014+0.0009 U/ml
(Mean.+-.Std Error).
Example 10
Establishing Baseline Response of Mice to Carbachol
[0170] C57BL/6 mice are used to establish the dose of Carbachol
that results in an at least a 2 fold increase of ERKI/II expression
over baseline level. Carbachol at doses of 0.15, 0.55 or 1.5
umol/Kg (36) are i.p. administered to 9-12 wk old female C57BL/6
mice, 5 mice/dose. 30 minutes post administration, the mice are
humanely euthanized and lung, brain and liver removed, protein
isolated and the level ERKI/II expression determined as described
herein. The dose of Carbachol (0.15 to 1.5 umol/Kg, more preferably
0.55 umol/Kg) that yields at least a 2 fold increase in ERKI/II
expression, and does not result in overt toxicity to the mice, is
desired.
Example 11
In Vivo Studies in Homozygous Negative BChE Knockout Mice
(BChE.sup.-/-)
[0171] As the homozygous negative knockout mice are virtually
devoid of BChE activity, they are a good model in which to confirm
the optimal molar ratio of pSCMV-mtBChE to pSCMV-ppro in the scL
complex, the dose to be administered and the resulting duration and
peak expression of the IN administered complex for in vivo use.
[0172] A total of 3 ratios surrounding and including the optimal
molar ratio of pSCMV-mtBChE to pSCMV-ppro determined in vitro are
assessed for BChE activity by the Ellman assay. The scL complex
prepared at the different ratios is administered via IN inoculation
of 9-12 wk old female BChE-/- mice (15 mice/group). Untreated mice
serve as controls. As the T.sub.1/2 of human plasma BChE is 11-14
days, 2 mice are euthanized from each group every other day from
day 0 (pre-treatment) today 14 and the level of BChE activity in
plasma at each time point assessed by the Ellman assay to determine
the time course and peak of activity. In addition, the relative
amount of tetramers, dimers and monomers in plasma is assessed via
non-denaturing gel electrophoresis as described herein to correlate
with activity. The ratio of between 10:1 and 1:10 (molar ratio of
inserts of pSCMV-mtBChE to pSCMV-ppro), more preferentially 5:1 to
1:5, most preferentially 4:1, that yields the longest lasting and
highest BChE activity is used to find the optimal in vivo DNA dose.
The optimal time of expression is between days 4 to 11.
[0173] Using the optimal ratio, the DNA dose IN administered via
the scL nanocomplex is varied. Initially, 3 doses are tested: 100,
75 and 50 ug total DNA. The scL-mtBChE/ppro complex, prepared as
described herein, is administered at the specified DNA dose via IN
inoculation of 9-12 wk old female BChE-/- mice (15 mice/dose).
Untreated mice serve as controls. The scL-mtBChE/ppro nanocomplex
is administered in a total volume of 50 to 400 .mu.L.
Unanesthetized mice are held along the back in the palm of the
hand. Holding the mice with the head tilted downward at an
approximate 30 degree angle, 10 uL of the nanocomplex is slowly
dripped into the nostril of the animal. The animal is allowed to
rest for a period of 30 to 60 seconds before an additional 10 .mu.L
is administered in the alternate nostril. This proceeds until the
entire solution is administered. For volumes over 100 uL, the
animals receiving the solutions are alternated after each 100 .mu.L
to permit more time between administrations for less stress on the
mice. At the peak expression time determined above, the animals are
humanely euthanized and the level of BChE activity in plasma with
each DNA dose determined by Ellman assay. In addition, the relative
amount of tetramers, dimers and monomers in plasma with each DNBA
dose are assessed via non-denaturing gel electrophoresis as
described herein. The optimal DNA dose gives at least 500 U/ml BChE
activity in plasma and results in at least 40%, more preferably
70%-90% tetramer form.
Example 12
Assess Activity of Exogenous mtBChe in BChE.sup.+/+ Mice
[0174] As this strain of mice is BChE.sup.+/+, this model is more
analogous to the human population. The optimal dose/ratio of the
DNA in the scL-mtBChE/ppro complex is administered via IN
inoculation of 9-12 wk old female C57BL/6 mice (20 mice/group) as
described herein. Untreated mice serve as controls. At the peak
expression time, the animals are humanely euthanized and the level
of BChE activity in plasma, determined by Ellman assay. In
addition, the relative amount of tetramers, dimers and monomers in
plasma is assessed via non-denaturing gel electrophoresis as
described herein. A level of BChE activity at least 2 fold greater
than the endogenous baseline level is desired.
Example 13
In Vivo Efficacy Studies
[0175] In these studies four groups of normal C57BL/6 mice are
used: Group 1=Untreated control; Group 2=Carbachol treatment alone;
Group 3=IN treatment with scL-mtBChE/ppro nanocomplex only; Group
4=IN treatment with scL-mtBChE/ppro nanocomplex followed by
Carbachol treatment. The optimal DNA dose in the scL complex at the
optimal ratio of the pSCMV-mtBChE to pSCMV-ppro vectors, (prepared
as described herein) is IN administered (as described herein) to
9-12 wk old female C57BL/6 mice (10 mice/group). At the time of
BChE peak expression, the animals in groups 2 and 4 receive (i.p.)
the optimal dose of Carbachol (0.15 to 1.5 .mu.mol/Kg, more
preferably 0.55 .mu.mol/Kg). Thirty minutes after Carbachol
administration, the mice are humanely euthanized and the level of
BChE activity in plasma assessed by the Ellman assay. ERKI/II
expression is also examined by Western analysis, in lung, brain and
liver tissues to correlate activity with down-modulation of
ERKI/II. Body weight of mice is measured to look for signs of
toxicity from the scL-mtBChE/ppro complex. Carbachol, a muscarinic
agonist, has been shown to stimulate muscarinic acetylcholine
receptor (mAChR) and activate ERKI/II, which have been shown to act
as a convergence site for various extracellular signals, including
mAChR activation. Prophylactic treatment with the scL complexes
suitably results in the inhibition of the Carbachol induced
expression of ERKI/II. A least 50% inhibition of ERKI/II is
desired. DNA dose in the scL complex is suitably modified (no
change in ratio) until it is achieved. Alternatively, multiple
administrations, every 3rd day to a total of 3 administrations,
prior to treatment with Carbachol can be given.
Example 14
Prophylactic Use of scL-mtBChE/Ppro as an Anti-OP Agent in Mice
[0176] The extent and duration of protection offered by IN
administered scL-mtBChE/ppro nanocomplex is determined in mice by
challenge with escalating doses of echothiophate (Wyeth-Ayerst),
which is a chemical warfare nerve agent-simulating compound.
Challenge experiments with echothiophate are performed using
wild-type strain C57BL/6 mice (BChE+/+) mice. scL-mtBChE/ppro at
doses of 50-100 .mu.g total pSCMV-mtBChE/pSCMV-ppro DNA (at the
preferred molar ratio of the inserts (10 to 1 to 1:10 [mtBChE to
ppro], more preferably 5:1 to 1:5, most preferably 4:1) is IN
administered as described herein. A group of mice do not receive
the scL-mtBChE/ppro nanocomplex. Plasma is collected from all mice
before IN administration and at the indicated intervals through day
11-21 post scL-mtBChE/ppro administration for assay of BChE
activity. On the day of peak activity (day 4 to 11) post IN
administration baseline temperature, body weights and observations
are recorded. Mice are challenged subcutaneously with
2.times.LD.sub.50 of echothiophate (200 ug/kg). Mice are observed
continuously through 1 h after echothiophate challenge. Axial body
temperatures and signs of cholinergic toxicity (straub tail,
hunched posture, and tremors) are observed periodically through
toxicant postchallenge. Moribund mice are euthanized immediately.
Animals that survive the first 2.times.LD.sub.50 challenge are
challenged with another 2.times. or 3.times.LD.sub.503 h after the
first challenge. Animals that survive the combined 4.times. or
5.times.LD.sub.50 doses of echothiophate are injected several hours
later with additional LD.sub.50 doses following the same
procedures.
[0177] Control mice are expected to die within a few minutes after
the first 2.times.LD.sub.50 dose of echothiophate, whereas animals
that receive scL-mtBChE/ppro are expected to survive a cumulative
dose of echothiophate of at least 2.times.LD.sub.50. The mice that
express the highest level of mtBChE in the plasma at the time of
the first challenge with echothiophate are expected survive the
highest level of echothiophate challenge. Correlation between
tolerated LD.sub.50 dose and plasma BChE levels at challenge are
expected.
Example 15
Use of scL-mtBChE/Ppro for Prophylaxis/Treatment Before and after
Exposure to OP Nerve Agents in Humans
[0178] The two pSCMV plasmid DNAs are mixed during preparation of
the nanocomplex at molar ratios (BChE to ppro) of the inserts of
10:1 to 1:10, more preferably 5:1 to 1:5, most preferably 4:1. The
nanocomplex is suitably made at ratios of 0.33 ug:10 ug:1 ug
(TfRscFv:Lip:DNA), with 5-20% Dextrose or Sucrose as an excipient
when HoKC is not a component of the complex; and at ratios of 0.3
mg:7 nmol:1 mg (TfRscFv:Lip-HoKC:DNA) with 5-20% Dextrose or
Sucrose when the complex comprises HoKC. The total amount of DNA in
the complex is 0.01 to 10 mg/kg/administration.
[0179] The size of the final complexes prepared by these methods
are suitably between about 50 and 500 (nm) with a positive zeta
potential (10 to 50 mV) as determined by dynamic light scattering
using a Malvern ZETASIZER.RTM. NANO-ZS. This size is small enough
to efficiently pass through the pulmonary epithelium and enter the
circulation and to cross the blood brain barrier.
[0180] The complex, prepared as above, is suitably used as either a
prophylactic or a therapeutic agent. For prophylaxis, the
ligand-targeted cationic liposome nanocomplex as prepared above is
administered into humans through a number of routes,
preferentially, inhalation, intranasal, oral, sublingual, etc. The
ligand-targeted cationic liposome nanocomplex as prepared above can
also be administered via other routes such as IM, IV, IP, ID
etc.
[0181] For prophylaxis, the nanocomplex can be self administered
once at least 6 hours, and suitably at approximately 24 to 48
hours, prior to exposure to a nerve agent to produce an effective
amount of BChE nerve agent neutralizing enzyme to protect against
at least 1.times.LD50, and up to 5.times.LD.sub.50, of the nerve
agent (including pesticides). It can also be self administered on a
regular basis (once or twice weekly) for at least 3 months prior to
any potential exposure to toxic nerve agents (including
pesticides). The preferred methods of self-administration are via
inhalation, intranasal, oral, sublingual, etc, but other methods of
self-administration such as IM, IV, IP, ID etc can also be
employed.
[0182] For therapeutic use, the scL-BChE/ppro nanocomplex can be
administered, either self-administered or administered by another
individual, anytime after exposure to nerve agents (including
pesticides) starting immediately post-exposure. The nanocomplex can
be administered in conjunction with other therapeutic agents and
can be repeatedly administered (as often as every 24 hours) as long
as medically necessary.
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[0220] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent or patent
application was specifically and individually indicated to be
incorporated by reference.
Sequence CWU 1
1
1125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptideMOD_RES(2)..(2)Histidylated
residueMOD_RES(6)..(6)Histidylated
residueMOD_RES(10)..(10)Histidylated
residueMOD_RES(14)..(14)Histidylated
residueMOD_RES(18)..(18)Histidylated
residueMOD_RES(22)..(22)Histidylated residue 1Lys Lys Lys Lys Lys
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys
Lys Lys Lys Lys Lys Cys 20 25
* * * * *