Note: Descriptions are shown in the official language in which they were submitted.
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NUCLEIC ACID DELIVERY FORMULATIONS
BACKGROUND OF THE INVENTION
This invention relates to methods and compositions for delivering nucleic
acids to
cells. In particular, the invention relates to delivery of nucleic acids for
the purpose of
gene expression from a bioabsorbable polymeric network structurally and
functionally
designed to induce gene expression.
Techniques for expression of exogenous DNA molecules hold considerable
potential for the treatment of hereditary diseases, e.g. cystic fibrosis.
These techniques
V
can also be used when expression of gene products from genes not naturally
found in the
' host cells is desired, for example, from genes encoding cytotoxic proteins
targeted for
expression in cancer cells. In one application, individuals can be treated
with an
exogenous DNA that can express a therapeutic polypeptide for some duration
(e.g., days,
weeks, a month, or several months) as needed for the particular treatment. DNA
vaccines
can be delivered in these formulations.
The emergence of methods for gene transfer to mammalian cells has prompted
enormous interest in the development of gene-based technologies for the
treatment of
human disease. To date, gene expression technology has focused primarily on
the use of
viral vectors that provide highly efficient transduction and high levels of
gene expression
iya vivo~ The most well-studied vectors are adenoviral vectors, particularly
those from
replication-defective viruses. These vectors can efficiently transduce non-
dividing cells,
generally do not integrate into the host cell genome, and can result in high
levels of
transient gene expression. However, the use of viral vectors has raised safety
issues
relating to, for example, host response to the virus, and oncogenic and
inflammatory
effects.
Other, non-viral gene transfer techniques that have been employed include
biolistic transfer, injection of "naked" DNA (US Patent No. 5,580,859),
delivery via
cationic liposomes (U.S. Patent No. 5,264,618), and delivery via
microparticles (U.S.
Patent No. 5,783,567), delivery via lipofection/liposome fusion products
(Proc. Nat'1
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Acad. Sci., Vol. 84, pp. 7413-7417 (1993), and methods based on the use of
polymers
that can be admixed with nucleic acids in solution and delivered to muscle
tissue (US
Patent 6,040,295).
A significant disadvantage of these methods is they usually provide for only
transient gene expression, and repeated administrations would thus be
necessary if
continued gene expression were needed.
SUMMARY OF THE INVENTION
The invention is based on the discovery that injectable and nucleic acid-
compatible polymeric compositions and formulations can be structurally
designed to
regulate gene expression i~ vivo~ for example, by controlling the
bioavailability of the
nucleic acid via modulation of the biodegradability and crosslink density of
the network
formed by the components of the formulation. The polymeric network encases the
nucleic acid, not only controlling the release of the DNA, but also providing
protection
from degradation. The invention described herein improves upon prior modes of
gene
delivery, in that gene expression can be regulated by modulation of a
polymeric network
formed by combination of at least two water-soluble components capable of
reacting with
one another. The nucleic acid of interest is incorporated into the network to
be released
in a sustained manner to achieve the level and duration of expression needed.
In general, the invention features an injectable aqueous formulation that
contains:
(a) a nucleic acid; (b) a first non-nucleic acid, water-soluble component; and
(c) a second
non-nucleic acid, water-soluble component, wherein the first and second
components
each include two or more reactive groups, the reactive groups of the first
component
being reactive with the reactive groups of the second component.
The first and second components of the formulation can react with one another
to
form a branched or a crosslinked polymeric network. The first and/or second
components can include one or more succinimidyl, chloroformate, acrylate,
amino,
alcohol, thiol epoxide, sulfhydryl, or hydrazidyl groups. In one example, at
least one of
the first and second components is a functionalized multi-armed poly(alkylene
oxide)
(i.e., a branched poly(alkylene oxide, or a poly(alkylene oxide) having more
than one arm
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(e.g., having eight or 16 arms emanating from a center) such as polyethylene
oxide),
polyethylene oxide)-co-poly(propylene oxide)-co-poly(ethylene oxide),
polypropylene
oxide)-co-poly(ethylene oxide)-co-poly(propylene oxide). In another example,
at least
one of the first and second components is a polyethylene glycol tetraamine. In
another
example, at least one of the first and second components is a polyethylene
glycol
tetrasuccinimidyl glutarate. In another example, at least one of the first and
second
components is a polyethylene glycol tetra-sulfhydryl. In another example, at
least one of
the first and second components is a functionalized poly(alkylene oxide) with
at least two
reactive functional groups, e.g., an epoxide, aldehyde, pyrophosphate, or any
other
functional group. In another example, at least one of the first and second
components is a
polyamidoamine having at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more
(e.g., 4 to 8 or
10 to 15) reactive functional groups, e.g., amino groups. In another example,
at least one
of the first and second components is a polyethylimine or polylysine
derivative. In
another example, at least one of the first and second components is a
functionalized
clutosan, cyclodextrin, or polyvinyl alcohol) with at least two reactive
functional groups.
In another example, one or both of the first and second components includes
three or
more reactive groups, the reactive groups of the first component being
reactive with the
reactive groups of the second component.
In one embodiment, a formulation of the invention can further include a third
non-
nucleic acid, water-soluble component. The third component can optionally
include at
least one reactive group. In some cases, the reactive groups) of the third
component can
be reactive with at least one reactive group of the first component or the
second
component, with both the first and second components, with a product formed by
reaction of the first and second components, or with neither the first nor the
second
component. In one example, the third component is methoxy-polyethylene glycol-
di-
stearoyl-phosphatidylethanolamine (PEG-DSPE).
In one embodiment, a formulation of the invention can further include an
excipient. In one example, the excipient is a neutral, anionic, or cationic
lipid. In another
example, the excipient is a sugar (e.g., sucrose, dextrose, or trehelose),
polyethylene
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glycol, chitosan, hyaluronic acid, chondroitin sulfate, heparan sulfate,
phosphatidyl
inositol, glucosamine, polyvinyl alcohol, Pluronics~ (BASF, Inc., Mount Olive,
North
Carolina, U.S.A.), derivatized Pluronics~, or derivatized polyethylene glycol.
In an example, an excipient includes a permeation enhancer. Examples of
"permeation enhancers" include pluronics (e.g., poloxamers), polyethylene
glycol,
polypropylene glycol, propylene glycol-based molecules, sodium dodecyl sulfate
(SDS),
poly-vinyl pyrrolidone (PVP), Vitamin E and Vitamin E-tocopherol acetate
(e.g., Vitamin
E-TPGS~, Eastman Kodak, Inc., Kingsport, Tennessee, U.S.A.), lauroyl and
oleoyl
macrogol glycerides (e.g., Labrafils~ and Gattefosse~, both available from
Gattefosse,
Westwood, New Jersey, U.S.A.), lipids, glycerol, polyoxyethylene sorbitan
monoesters,
Tween~ 20 and 80, Span~ 80, fatty acids, fatty acid esters, bile salts (e.g.,
taurocholic
acid and glycocholic acid), Brij~, sodium hyaluronate (Genzyme Corp,
Framingham,
MA, USA), bolaphiles, and sorbitan oleates (Sigma, Inc.).
In another example, the excipient includes a bioavailability enhancer.
Examples
of "bioavailability enhancers" include propylene glycol and macrogol-based
enhancers
(e.g., Gelucire~ (Gattefosse), Labrafil~ (Gattefosse), Capryol~ (Gattefosse),
Labrasol~
(Gattefosse), Plurol~ (Gattefosse)), Bioperine~ (Sabinsa Corporation, New
Jersey,
U.S.A.), Vitamin E (Sigma, Inc.)and Vitamin E- TPGS~ (Eastman Kodak),
poloxamers
such as Pluronics~ (BASF, Inc.), and polyethylene glycol (Sigma, Inc.).
In another example, the excipient is a protein (e.g., contains a cytokine).
In another example, the excipient contains a small molecule drug, e.g., an
anti-
tumor agent, anti-neoplastic, anti-inflammatory, or antibiotic.
In another example, the excipient is an adjuvant (e.g., a CpG oligonucleotide,
oil,
lipid, monophosphorolipid (MPL; Sigma, Inc.), lipopolysaccharide(LPS; Sigma,
Inc.), or
carbohydrate).
In another example, the excipient is chemically bound to the crosslinlced
polymeric network or branched polymer, e.g., methoxy PEG-monoamine,
distearoylethanolamine, stearylamine, spermine, spermidine, laurylamine, urea,
dioleylethanolamine, or aminocaproic acid. All of these excipients are
reactive with the
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network, forming covalent bonds. In another example the excipient contains a
component
that stabilizes a nucleic acid, e.g., sodium, calcium, zinc, or magnesium
salts of
bicarbonates.
An example of a reactive excipient is phosphatidyl ethanolamine, which can
react
with polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG). Another example
of a
reactive excipient is a poly(amino acid) containing multiple cysteines in its
backbone
(e.g., poly(cysteine) or a peptide susch as poly(arg-lys-cys-guanine-arg-cys-
cys-lys-cys)).
The free -SH of the cysteines can would react easily with P4-SG. Another
example is
poly(lysine), with the pendant amino groups of which can reacting easily with
P4-SG.
When P4-SG and/or polyethylene glycol)-tetraamine (P4-AM) are components of
the
new formulations, Just cysteine or lysine can also be used as an excipient.
In another aspect, the invention features an injectable aqueous formulation
that
contains: (a) a nucleic acid; (b) a first non-nucleic acid, water-soluble
component; (c) a
second non-nucleic acid, water-soluble component, and (d) a third non-nucleic
acid,
water soluble component, wherein the first, second and third components each
include
two or more reactive groups, the reactive groups of the third component being
reactive
with the reactive groups of the first component or the second component.
A formulation can include more than one species of nucleic acid, e.g., two or
more species of nucleic acids, each encoding a different polypeptide or a
nucleic acid
encoding a polypeptide and an oligonucleotide. In addition, a nucleic acid can
be an
oligonucleotide (e.g. with a phosphodiester or phosphorothioate backbone). In
one
example, a nucleic acid encodes a therapeutic protein or a protein that
induces an immune
response. As used herein, a "therapeutic protein" is a protein that when
administered to
an individual confers a therapeutic benefit upon the individual. By "protein
that induces
an immune response" is meant a pathogenic protein (e.g., a viral or bacterial
protein) or
portion thereof, a tumor-associated antigen or portion thereof, or another
protein that is
involved in disease (e.g., a neurodegenerative (e.g., Alzhiemer's), cardiac,
immunologic,
autoimmune, or gerontologic disease).
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A nucleic acid of a formulation described herein can be in any form, e.g., a
solution, dispersion, powder, precipitated, condensed, micronized, or
emulsion. A
nucleic acid can optionally be encapsulated in or associated with a
biodegradable
polymeric microparticle. Examples of useful microparticles are described in
U.S. Patent
5,783,567, USSN 09/909,460 (which is a continuation of USSN 09/321,346), and
USSN
091872,836, the contents of which are incorporated by reference in their
entirety. In one
example, the nucleic acid is released from the branched or crosslinked
polymeric network
by biodegradation or by simple diffusion.
In one embodiment, a formulation described herein forms a hydrogel at a
temperature between about 20°C and about 40~C within about 20 minutes
after the
formulation is prepared. In other embodiments, a formulation described herein
forms a
hydrogel at a temperature between about 25, 30, 35, or 37~C and about 40~C,
within
about 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or
less than 1 minute
after the formulation is prepared. In one example, a formulation described
herein
remains injectable for at least fifteen seconds, e.g., at least 15, 30, 45,
60, 90, 120, 180,
240, 300, or 600 seconds, or 15 minutes or 20 minutes after the formulation is
prepared.
In another embodiment, the network of a formulation described herein forms a
viscous liquid.
In another embodiment, the nucleic acid is protected from serum nucleases by
incorporation into the network. In one example, the nucleic acid is expressed
following
injection of the network (e.g., into muscle). In another example, an immune
response is
generated to the nucleic acid encoded antigen following injection of the
networl~/nucleic
acid formulation. In another example, the release of the nucleic acid
following injection
is controlled by the cross-linking density of the network. In another example,
the
expression of the nucleic acid following injection is controlled by the cross-
linking
density of the network.
The first and/or second components of a formulation can be biodegradable,
e.g.,
by a hydrolytic or proteolytic mechanism. The network of formulation can be
biodegradable, e.g., by a hydrolytic or proteolytic mechanism.
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The branched or crosslinked polymeric network, e.g., fully or partially
crosslinked, of a composition can include linkages selected from the group
consisting of
ester, carbonate, imino, hydrazone, acetal, orthoester, peptide, amide,
urethane, urea,
amino, oligonucleotide, and sulfonamidyl bonds. As used herein, "partially"
means that
the stoichiometry of the components can be adjusted, so that some of the
functional
groups remain unreacted, e.g., to obtain a loose network. As used herein,
"fully" means
that the stoichiometry of the components is equimolar, e.g., essentially all
available
functional groups have been reacted in the network.
The first andlor second components of a formulation can include one or more
sulfhydryl, amine, epoxide, phosphoroamidates, chloroformate, acrylate,
carboxylic acid,
aldehyde, succinimide ester, succinimide carbonate, maleimide, iodoacetyl,
carbohydrate,
isocyanate, and/or isothiocyanate groups.
At least one of the first and second components of a formulation described
herein
can include a biodegradable linkage such as a lactate, caproate, methylene
carbonate,
glycolate, ester-amide, ester-carbonate, or a combination thereof.
In another embodiment, the formulations described herein can be in the form of
microparticles (e.g., microparticles, nanoparticles, microspheres, or
nanospheres). Such
microparticles can be essentially "solid," meaning that the cross-linked
polymer (e.g., the
hydrogel) formed by reaction of the first and second non-nucleic acid
components can be
distributed, evenly or unevenly, throughout each microparticle, with the
nucleic acid
distributed within the three-dimensional structure of the polymer.
Alternatively, the
microparticles can have outer shells made up of the cross-linked polymer, and
the nucleic
acid can be either within the polymeric structure or else in the core of the
microparticle.
The invention also features a method for making such microparticles. The
method
includes introducing the nucleic acid and the first and second non-nucleic
acid
components of any of the formulations described herein into an emulsifying
bath (e.g., a
homogenizer or blender, or other device capable of emulsifying a mixture),
either
separately or after combining, and then emulsifying (e.g., by homogenizing or
blending)
the resulting mixture in the emulsifying bath for at least part of the time
that the first and
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second non-nucleic acid, water-soluble components are reacting with each
other. By
adjusting the concentrations, ratios, emulsification speed, and identities of
the
components, the size, structure, and other physical properties of the
microparticles can be
controlled. For example, microparticles smaller than about 500 microns, 250
microns,
100 microns, 50 microns, 20 microns, 15 microns, 10 microns, 5 microns, 2
microns, 1
micron, or still smaller can be prepared. Generally, higher homogenization
rates result in
smaller microparticles.
In another aspect, the invention includes a method of making'a polypeptide by
applying a formulation described herein to a cell. The nucleic acid contained
within the
formulation can code for expression of the polypeptide. In one example, the
formulation
. is applied to a cell within an animal, e.g., administered to the animal by
injection,
extrusion, or spraying.
In another embodiment, the invention includes a method of making a polypeptide
by injecting into an animal, e.g., a mouse, rat, pig, non-human primate, or
human, a
formulation described herein. The nucleic acid contained within the
formulation can
code for expression of the polypeptide. In one example, the formulation is
injected in,
on, or adjacent to a tumor. In another example, the formulation is injected
intramuscularly, subcutaneously, or intra joint. The formulation can be
injected into the
animal once or more than once. The formulation can be delivered, for example,
via an
aerosolizer or nebulizer. The formulation can alternatively be applied to the
skin,
delivered in a patch, or placed on a wound. The formulation is also suitable
for delivery
via needle-free devices. The formulation can be delivered by any of these
mechanisms
and then followed by an electrical pulse. Electrical pulses are known to
enhance uptake
of macromolecules post injection as described in U.S. Patent No. 5,993,434.
The
formulation can be premixed before injection.
In another aspect, the invention includes a method of producing a polypeptide
by:
(a) providing a surface suitable for cell culture; (b) adding a formulation of
the invention
to the surface; and (c) placing a cell on the formulation. According to this
method, the
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nucleic acid codes for expression of a polypeptide, and the cell produces the
polypeptide
following the culturing of the cell iya vitf°o~
In another aspect, the invention features a method of making a dried nucleic
acid
formulation by: (a) preparing a mixture by mixing in an aqueous solution (i) a
nucleic
acid, (ii) a first non-nucleic acid, water-soluble component, (iii) a second
non-nucleic
acid, water-soluble component, and (iv) a third non-nucleic acid, water-
soluble
component, wherein the first and second components each include two or more
reactive
groups, the reactive groups of the first component being reactive with the
reactive groups
of the second component at a pH greater than 7Ø Optionally, the third
component can
include at least one reactive group that is reactive at a pH greater than 7.0
with at least
one reactive group of the first component, the second component, of both the
first and the
second components, of a product formed by reaction of the first and second
components,
or with neither the first nor the second component. The aqueous solution has a
pH and/or
temperature that prevents the first and second components from reacting to
form a
crosslinked network (i.e., at a pH lower than 7.0); and (b) drying the mixture
to thereby
create a dried formulation (e.g., drying in a lab dryer under vaccum, or in a
lyohilizer).
This method permits the preparation of a formulation that contains unreacted
components
in a single vessel, e.g., the method avoids the necessity of maintaining the
components in
separate vessels until just prior to initiating a crosslinking reaction. The
mixing step~can
be performed at a pH less than about 7.0, e.g., less than about 6Ø In one
example, the
mixing step is performed at a pH of about 5.5. The mixing step can be
performed at or
below about 4°C, e.g. between 0°C and 4°C. In one
example, the miXture is dried. In
another example the mixture is lyophilized.
In another embodiment, both components can be individually dried (optionally
with nucleic acid and/or excipient in one or both of the components), and then
a buffer is
added to reconstitute the formulation.
In one embodiment, the first non-nucleic acid, water-soluble component is
polyethylene glycol amine. In another embodiment, the second non-nucleic acid,
water-
soluble component is polyethylene glycol succinimidyl glutarate. In another
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embodiment, the first non-nucleic acid, water soluble component is
polyethylene glycol
sulfhydryl. In another embodiment, the third non-nucleic acid, water-soluble
component
is methoxy-polyethylene glycol-di-stearoyl-phosphatidylethanolamine (PEG-
DSPE).
The invention also features a method of preparing a gel-forming nucleic acid
formulation. The method entails adding a buffer having a pH greater than 7.0
to a dried
nucleic acid formulation of the invention. The addition of the buffer results
in the
formation of a crosslinked network between the first and second components.
For
example, the buffer can be a phosphate buffer with a pH of about 7.5. The
buffer can
include nucleic acid and/or excipients (e.g., sucrose, Tris, EDTA). The adding
step can
be performed at or above 20°C, e.g., at or above 37°C.
In another aspect, the invention features a dried nucleic acid formulation
that
contains: (a) a nucleic acid; (b) a first non-nucleic acid, water-soluble
component; (c) a
second non-nucleic acid, water-soluble component; and (d) a third non-nucleic
acid,
water-soluble component. The first two components, and optionally the third
component
as well, each include two or more reactive groups, the reactive groups of the
first
component being reactive with the reactive groups of the second component or
with the
reactive groups of the third component, or both, and/or the third component
also includes
at least one reactive group that is reactive with at least one reactive group
of the first
component, with at least one reactive group of the second component, with at
least one
reactive group of each of the first and second components, with a reactive
group of the
product formed by reacting the first and second components, or with neither
the first nor
the second component. Tn the dried formulation, the three components are each
in an
unreacted state, and the nucleic acid and the three components are not in
solution. The
formulation can be lyophilized. In one embodiment, the first non-nucleic acid,
water-
soluble component is polyethylene glycol amine. In one embodiment, the first
non-
nucleic acid, water-soluble component is polyethylene glycol sulfliydryl . In
still another
example, the second non-nucleic acid, water-soluble component is polyethylene
glycol
succinimidyl glutarate. In another embodiment, the third non-nucleic acid,
water-soluble
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component is methoxy-polyethylene glycol-di-stearoyl-phosphatidylethanolamine
(PEG-
DSPE).
The invention also features a kit containing a dried formulation described
herein;
and a buffer having a pH of at least 7Ø
The invention also includes a method of administering a nucleic acid to an
individual by: preparing a mixture by adding a buffer having a pH of at least
7.0 to a
dried formulation described herein; incubating the mixture to permit the
formation of a
crosslinked network; and administering the mixture to the individual.
As used herein, a "nucleic acid" can be either RNA or DNA, including, for
example, cDNA, genomic DNA, oligonucleotides, mRNA, viral DNA, bacterial DNA,
plasmid DNA, triplex nucleic acid, peptide-nucleic acid (PNA) formulations, or
condensed DNA. In a preferred embodiment, the nucleic acid is plasmid DNA. In
another embodiment, the nucleic acid is an oligonucleotide. The
oligonucleotide can
include stabilizing features such as base or backbone modifications (e.g.,
phosphorothioate backbone). The oligonucleotide can be an antisense
oligonucleotide,
utilized to treat various diseases. In one example, the oligonucleotide can
have anti-
tumor activity. In yet another example, the oligonucleotide can be used as an
adjuvant,
e.g., as described in EP 01005368 and WO 99161056.
By "bioavailability of the nucleic acid" is meant that the delivery
formulation
prolongs availability of the nucleic acid. By altering the polymer
formulation, one can
increase or decrease the rate of release of nucleic acid from the polymer
netwoxk, in turn
affecting activity or expression levels. In some embodiments, the polymeric
network is
biodegradable, i.e., it breaks down into components that are readily cleared
from the
body.
By "modulation of the polymeric network" it is meant, for example, that the
hydrolytically labile linkages can be varied in length and type to affect the
degradation
time of the network. A fast-degrading polymeric network would provide a higher
bioavailability of the nucleic acid to the target cells than would a slower
degrading
network. Alternatively, excipients can be added to enhance breakdown of the
network.
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For example, succinimidyl propionates, succinimidyl caproate or succinimidyl
carbonates
can be substituted for succinimidyl glutarate in a PEG component to lower the
rate of
hydrolytic degradation of the network. Conversely, sulthydryls can be
substituted for
amines in a PEG component to increase the rate of hydrolytic degradation of
the network.
In another example of modulation of the polymeric network, the concentration
of
the gel-forming components can be varied to change the nature of the network.
Thus, for
example, a higher concentration of these components results in longer
degradation times
and increased branching and/or cross-linking, leading to a lower availability
of the
nucleic acid to the target cells and, consequently, a lower expression level
or a lower
level of therapeutic nucleic acid.
In still another example of modulation of the polymeric network, the molecular
weight of the gel-forming components can be selected to vary the nature of
network,
particularly the molecular weight between cross-links. A tighter network can
result from
the use of lower molecular weight components, causing greater retention of the
DNA at
the site and, consequently, sustained release for a longer duration. By
"sustained
release", it is meant that the nucleic acid is available to the target cells
for uptake for a
longer period of time than would be achieved if the administration of the
nucleic acid
were in, for example, saline, from which fast dissipation of the DNA from the
site would
occur.
In yet another example of modulation of the polymer network, addition of a
third
polymer to one of the pre-formulation components, to form either
an.interpenetrating
network or a semi-interpenetrating network, can vary the nature of the network
to control
release of DNA at the site to control level and duration of expression.
Examples of
suitable "third polymers" include methoxypolyethylene oxide-monoamine,
polyethylene
glycol, poloxamers, and methoxypolyethylene oxide-distearoyl ethanolamine and
8-, 16-,
and 32-arm derivatized and non-derivatized polyethylene oxide. In another
embodiment,
the invention features a method of delivering a particle to an individual. The
method
includes administering to the individual a formulation that includes: (1) the
particle; (2) a
first non-nucleic acid, water soluble component; and (3) a second non-nucleic
acid, water
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soluble component. The first and second components each include two or more
reactive
groups, the reactive groups of the first component being reactive with the
reactive groups
of the second component. The particle can be, for example, a virus or viral
particle or a
virus-like particle (VLP) (e.g., adenovirus or adenoviral particle such as an
aviadenovirus
or mastadenovirus or a penton, hexon, capsid, or other fragment thereof, or
VLP made of
hepatitis, or papillomavirus components).
By "excipient" is meant a molecule added for the purpose of enhancing or
sustaining DNA uptake, activity, or expression, or to further enhance DNA
stability, or to
modulate release of DNA or degradation of the network. In certain embodiments,
the
excipient is a bioavailability enhancer. By "bioavailability enhancer" is
meant an
excipient that improves or enhances bioavailability of the DNA to the target
cells by its
retention at the cell site.
The invention provides several advantages. For example, the new methods and
formulations feature an injectable polymer-based slow release system that can
afford
sustained systemic protein expression (e.g., by delivering genes into skeletal
muscles).
Such a system can be used in the treatment of "chronic" diseases where
multiple
administrations are necessary to maintain therapeutic levels of bioactive
proteins and
peptides. Sustained gene delivery can in turn allow for long-term protein
expression. Iy
vitro release experiments described herein indicate that plasmid DNA is slowly
released
over time from the crosslinked network formulations, with higher crosslinlced
hydrogels
releasing the DNA more slowly. The formulated plasmid DNA generated longer-
term
protein expression compared to unformulated "naked" DNA in both
immunocompetent
and complement deficient animals.
The new formulations can also provide protection to the entrapped DNA.
Combined with plasmid stability, the new formulations can significantly
increase the
duration of protein expression following a single administration of DNA;
genetic
approaches can generate longer protein expression since the intracellular half
life of
plasmid DNA is generally much longer than the serum half life of recombinant
proteins.
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The expression kinetics can be further prolonged by slowly releasing the
plasmid over
time so that the source of the protein is bio-available for several weeks.
Another advantage of the new formulations of the invention is that they are
injectable. Polyethylene glycols are considered to be biomimetic and hence
highly
biocompatible. They have also been shown to generate minimal inflammation and
immune response. Moreover, the new formulations are injectable following
reconstitution and do not require surgical implantation procedures.
Furthermore, the crosslinked networks of the invention are readily
biodegradable
due to the presence of, for example, hydrolytic ester linkages on the P4-SG
component.
The network components can have a molecular weight on the order of about
10,000
I~altons; upon degradation the components can be cleared from the body quite
readily.
Unless otherwise defined, all technical and scientific terms used herein have
the
same meaning as commonly understood by one of ordinary skill in the art to
which this
invention belongs. Although methods and materials similar or equivalent to
those
described herein can be used in the practice or testing of the present
invention, the
preferred methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are incorporated
by reference
in their entirety. In case of conflict, the present application, including
definitions, will
control. In addition, the materials, methods, and examples are illustrative
only and not
intended to be limiting.
Other features and advantages of the invention will be apparent from the
following detailed description, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a chemical structure of certain network components and schematic
representation of the crosslinking reaction. The amine and succinimidyl groups
react to
generate amide linkages between the polymer species thereby forming the
network
structure. The hydrolytically labile ester linkages in the P4-SG render the
network
biodegradable.
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FIGS. 2A to 2C are graphs depicting network characterization by gel permeation
chromatography (GPC) and viscometry. FIG 2A is a graph of GPC analysis of
formulation A. Individual PEG components (P4-SG and P4-AM) are indicated by
arrows
as is the resulting network, FIG 2B is a graph of formulations A, B, C and D
that were
analyzed by viscometry. Data were collected and plotted at different intervals
after
mixing the two PEG components using the Brookefield WingatherTM software.
'Tiscosity
was measured at 37°C (formulation A) and 25°C (formulations B, C
and D), FIG 2C is a
graph of gelation time (y-axis) plotted as a function of gel concentration (x-
axis).
FIG. 3 is a table summarizing the physico-chemical characteristics of network
formulations A (2% w/v P4-AM/P4-SG), B (3% w/v P4-AM/P4-SG)~ C (4% w/v
P4-AM/P4-SG) and D (5%w/v P4-AM/P4-SG), detailing appearance of gels, gel
swelling
and gelation times determined at 25°C and 37°C.
FIG. 4 is a picture of a gel showing chemical compatibility of pDNA with
network components. In lane 1 is a secreted embryonic alkaline phosphatase
(SEAP)
plasmid, in lane 2 is 1 p,g/ml of DNA incubated with 2% w/v (P4-AM+P4-SG);and
in
lane 3 is 1 ~g/ml of DNA incubated with 5% w/v (P4-AM+P4-SG).
FIGS. 5A and SB are two plots of the swelling properties of 5% (grey), 8%
(black), and 10% (white) PEG hydrogels formulated at different intervals
following stock
solution preparation to examine solution stability. Overnight swelling
(percent increase
in weight) was performed at 37°C in phosphate-buffered saline (PBS)
with blank PEG-
hydrogels (A) or with hydrogels containing plasmid and mPEG-DSPE (B).
FIG. 6 is a table depicting the injectability of P4-AM/P4-SG formulations.
Maximun time for injection (min) after reconstitution is shown for
formulations A (2%
w/v P4-AM/P4-SG), B (3% w/v P4-AM/P4-SG), C (4% w/v P4-AM/P4-SG) and D (5%
w/v P4-AM/P4-SG).
FIG. 7A is a graph showingin vitro release of plasmid from network
formulations
as measured by HPLC analysis. Depicted is a typical HPLC trace of DNA released
from
formulation C. The first peak represents polyethylene glycol, the second set
of peaks
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(triplet) represents different isoforms of plasmid (supercoiled plasmid is
represented by
the second peak).
FIG. 7B is a graph depicting cumulative release of DNA from formulations B, C,
and D at different time intervals (days post administration). Day 1 is
represented by the
white bar, day 3 by the black bar, day 7 by the stippled bar, and day 14 by
the grey bar.
FIG. 8 is a picture of a gel depicting protection of network entrapped DNA
from
serum digestion. In lane 1 is unformulated DNA, no serum; in lane 2 is
formulation A,
no serum; in lane 3 is formulation B, no serum; in lane 4 is unformulated DNA
+ 1:40
serum; in lane 5 is formulation A + 1:40 serum; in lane 6 is formulation B +
1:40 serum;
in lane 7 is unformulated DNA + 1:80 serum; in lane 8 is formulation A + 1:80
serum;
and in lane 9 is formulation B + 1:80 serum. The arrow represents supercoiled
DNA.
FIG. 9 is a graph depicting the swelling properties of 10% w/v P4-SG/0.5% w/v
poly(amidoamine) (PAMAM) hydrogels. Swelling was tested following overnight
incubation of gels at 37~C (n=3). Gels were formed in the presence (w/mPEG-
DSPE) or
absence (w/o mPEG-DSPE) excipient.
FIG. 10 is graph depicting the analysis of gel times for P4-SGlpoly(ethylene
oxide)-sulfydryl (P4-SH) networks. Viscosity was measured at 25~C for 3%, 4%,
and
10% w/v PEGs formulations. Symbols for each gel formulation are indicated. Y-
axis
represents viscosity (cp) and the x-axis represents time (minutes). Data were
collected at
different intervals after mixing the two PEG components using the Brookefield
WingatherTM software.
FIGS. 11A and 11B are graphs depicting the expression of SEAP in mice injected
with network containing SEAP DNA. FIG 11A shows serum SEAP level indicated on
the y-axis (ng/ml) and the formulation is indicated on the x-axis. Time points
are
indicated by different filled bars. FIG 11B shows the percent of animals
within each
group that express more than 300 pg/ml serum SEAP (y-axis) at days 10 (black
bars), 33
(striped bars), and 92 (white bars), as indicated for each formulation (x-
axis).
FIGS. 12A and 12B are graphs depicting the SEAP expression in complement
deficient DBA/2 mice. FIG 12A show the percent of SEAP expressing animals
(animals
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expressing >300 pg/ml at a given time point) as indicated on the (y-axis).
Mice were
injected with the SEAP DNA containing formulation groups indicated on the (x-
axis).
Timepoints are as indicated (days 7, 35, 81). FIG 12B shows expression of
serum SEAP
in RAG2 immunocompromised mice injected with P4-AM/P4-SG networks containing
SEAP plasmid DNA. Percent of SEAP expressing animals (animals expressing >300
pg/ml at a given time point) is indicated on the (y-axis). Mice were injected
with the
SEAP DNA containing formulation groups indicated on the (x-axis). Timepoints
are as
indicated (days 7, 14, 30 and 42).
FIG. 13 is a graph depicting the effect of electroporation on serum SEAP
levels.
Mice were injected with a GT20 P4-AM/P4-SG formulation. Half the animals
received
electroporation treatment ("+EP") as depicted on x-axis. Serum SEAP level is
indicated
on the y-axis (ng/ml). Serum samples were tested 7 days post administration in
mouse
muscle.
FIGS. 14A and 14B are graphs depicting how serum SEAP expression can be
influenced by network containing excipients. FIG 14A shows serum SEAP levels
(ngs/ml) as indicated on the y-axis. Excipients formulated with the GT20
network are
indicated on the x-axis. The bars represent the following excipients
formulated with
GT20 networks, respectively: GT20 + 0.1% SDS, GT20 + 0.1% L62, GT20 + 0.15%
PAMAM. FIG 14B is a graph showing GT20 + 0.025% w/v Streptolysin, GT20 +
250mg/ml, Magainin I, GT20 with no excipient. Serum samples were tested 7 days
post
administration in mouse muscle.
FIG. 15 is a graph depicting how SEAP expression is mediated by DNA in
P4-SH/P4-SG networks. Serum SEAP levels (ngs/ml) are indicated on the y axis
and the
formulation is indicated on the x-axis (3.5 %w/v, formulation A; 5 %w/v,
formulation B).
Serum samples were tested 7 days post administration.
FIG. 16 is a graph depicting how ~3-gal specific antibody is elicited in mice
immunized with network formulated DNA. Titers of ~-gal specific IgG from
pooled
serum samples (n=4) were determined 12 weeks post injection. Titers from
individual
serum are indicated on the y-axis and the formulation is indicated on the x-
axis. The data
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are presented as the mean -~- standard error (SE) of four mice performed in
duplicate. *,
p=0.014 formulation A vs saline control; **, p<0.01 formulation B vs saline
and p=0.137
formulation A vs formulation B by Student's t test.
FIG. 17 is a graph depicting T cell proliferative ~3-gal specific responses in
mice
immunized with formulated DNA. Responses are from pooled samples (n=4) at 12
weeks post-immunization. Antigen used in the stimulation is indicated (~3-gal
(dark bars)
or chicken ovalbumin (OVA) (white bars) protein). The immunizing formulation
is
indicated on the x-axis and the stimulation index (SI, the median counts per
minute (cpm)
of the maximum response to antigen divided by the median cpm in the absence of
antigen) is indicated on the y-axis. Data are expressed on the y-axis as mean
of triplicate
samples ~ SE. *, p=0.01 formulation A vs saline; **, p<0.001 formulation B vs
saline
and p=0.26 in comparison between two formulations.
FIG. 18 is a graph depicting interferon gamma Elispot analysis of T cells in
mice
immunized with network formulated DNA. Splenic T cells were harvested at at 12
weeks post-immunization and pooled (n=4). Response to H-2Ld restricted, ~3-gal
87(-
gg4 peptide (filled bars) or HBV peptide (hatched bar) or media (open bar) is
indicated.
The number of IFN-Y + spot forming cells/10~ T cells is indicated on the y-
axis. The
relevant formulation (A or B) and untreated control are indicated on the x-
axis. The data
are presented as the mean ~ SE of four mice performed in triplicate. *,
p<0.001 in
comparison with saline control; p=0.52 formulation A vs formulation B.
FIG. 19 is a table depicting protection of mice immunized with network
formulated DNA. BALB/c mice were challenged by i.v injection of either 5 x 10~
CT26.WT or ~-gal expressing CT26.CL25 tumor cells, three weeks post
immunization.
The number of tumor nodules is indicated in each group.
FIG. 20. is a schematic depicting a method of preparing a lyophilized
formulation
that can be reconstituted in a single vial prior to use.
FIG. 21 is a graph depicting how lyophilization does not effect gel time.
Viscosity of a 10% w/v P4-SH/P4-SG network formulation that was not-
lyophilized
compared to a reconstituted lyophilized formulation. Symbols for each
formulation are
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indicated. The y-axis represents viscosity (cp) and x-axis represents time
(minutes).
Data were collected at different intervals after mixing the two PEG components
using the
Brookefield WingatherTM software.
FIG. 22 is a graph depicting interferon gamma Elispot analysis of T cells in
mice
immunized with lyophilized or non-lyophilized formulations. Formulations
included 2%
w/v P4-AM/P4-SG and 3% w/v P4-AM/P4-SG. Splenic T cells were harvested at at
12
weeks post-immunization and individually analyzed (n=4). Mean responses to H-
2Ld
restricted, (3-gal g7(-gg4 peptide (filled bars) or HBV peptide (hatched bar)
or media
(open bar) are indicated: The number of IFN-Y spot forming cells/106 T cells
is indicated
on the y-axis. The relevant formulations administerd as reconstituted
lyoplulized or non-
lyophilized formulations, and a saline control are indicated on the x-axis.
p<0.001 in
comparison with saline control; p=0.156 for 3% lyophilized vs unlyophilized
formulations and p=0.137 for 2% lyophilized and non-lyophilized formulations.
FIGS.23A and 23B are graphs depicting release of oligonucleotide from
P4-SG/P4-AM gels. Release assays were performed for oligonucleotide in 5% w/v
(a)
and 10% w/v formulations (b) (x-axis). Release was carried out in phosphate
buffered
saline, pH 7.4 at 37~C, with n=3 per timepoint. Percent of oligo released
within each time
frame is indicated on the y-axis.
FIGS. 24A and 24B are graphs depicting how viscosity of a 10 % w/v P4-SH/P4-
SG network formulation varies with temperature and pH. The y-axis represents
viscosity
(cp) and the x-axis represents time (minutes). In FIG 24A, viscosity was
performed at
25°C or 37~C as indicated. In FIG 24B, viscosity measurements were
performed at
various pHs as indicated. Data were collected at different intervals after
mixing the two
PEG components using the Brookefield WingatherTM software.
FIGS. 25A and 25B are graphs depicting oligonucleotide release from P4-SH/P4-
SG gels. The release study was carried out in phosphate buffered saline, pH
7.4 at 37~C,
with n=3 gels per timepoint. In FIG 25A, the y-axis indicates percent oligo
released and
the x-axis represents the time frame. In FIG 25B, cumulative oligonucleotide
release (y-
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axis) is plotted versus time (x-axis). Release was performed on 10, 20 and 30%
gels as
indicated.
FIG. 26 is a graph depicting oligonucleotide release from 10% w/v P4-
SG/PAMAM, GO gels. The release study was carried out in phosphate buffered
saline,
pH 7.4 at 37~C, with n=3 gels per timepoint. The y-axis indicates percent
oligo released
and the x-axis represents the time frame.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to methods and compositions for delivering nucleic
acids to
cells. These methods and compositions can be used for a variety of functions
including
but not limited to the induction of cell activation, the regulation of gene
expression, or the
induction of gene expression. A nucleic acid is released from a bioabsorbable
polymeric
network structurally and functionally designed to enhance and optimize the
level and
duration of the released nucleic acid activity or expression.
The composition of the delivery system includes a polymeric network formed by
the chemical combination of at least two injectable non-nucleic acid polymeric
components, containing one or more nucleic acids and one or more excipients.
The components (l, 2, and optionally 3) are water-soluble and are composed of
polymeric backbones modified to have end functional groups capable of reacting
with
one another. The reactive functional groups of component 1 can be, for
example,
chloroformates, acrylates, amines, alcohols, tetrasulfydryls, epoxides;
sulfhydryls,
hydrazides, or combinations thereof, in the same molecule. The reactive
functional
groups of component 2 can be, for example, chloroformates, acrylates,
carboxylic acids,
aldehydes, maleimides, iodoacetyl, carbohydrates, isocyanates, or
isothiocyanates. The
polymeric network can include linkages such as esters, carbonates, imines,
hydrazones,
acetals, orthoesters, peptides, amides, urethanes, areas, amines,
oligonucleotides, or
sulfonamides. The components can be modified to include biodegradable linkages
such
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as lactates, caproates, methylene carbonates, glycolates, ester-amides, ester-
carbonates, or
combinations thereof.
The following are examples of the practice of the invention. The examples
demonstrate examples of various polymer networks for formulation,
characterization and
modulation to optimize gene expression levels and duration. They are not to be
construed as limiting the scope of the invention in any way.
EXAMPLES
Example 1: Iu_situ Formation of Polyethylene Oxide-Polyethylene Oxide
Networks via Formation of Amide Linkages
Reacting Polymers
Component 1: Polyethylene oxide-tetraamine (P4-AM), (SunBio Systems,
Korea)
Component 2: Polyethylene oxide 3350)-tetrasuccinimidyl glutarate (P4-SG),
(Sunl3io Systems)
Polymer Characterization
The degree of substitution (d.s.) of amines on the tetra-armed polyethylene
oxide
backbone was calculated to be 3.91 by 1H-NMR; d.s. of succinimidyl glutarate
was 3.85,
also by 1H-NMR.
Preparation of Formulations
All formulations were prepared by mixing of two solutions, one containing a
pre-
weighed amount of P4-AM dissolved in O.1M potassium phosphate buffer, pH 8.0
and
the other containing an equimolar amount of P4-SG dissolved in cold deionized
water
containing nucleic acid (e.g. plasmid or oligonucleotide). All formulations
contained 1
mg/ml of the nucleic acid. FIG. 1 shows the chemical structure of the network
components (P4-AM/P4-SG) and a schematic representation of the cross-linking
reaction
by formation of amide linkages. The netyvork is rendered biodegradable by the
presence
of ester linkages in one of the components, P4-SG.
Formulation of 2%-15% Polymeric Networks
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Solutions of the geI-forming components were prepared (2%, 3%, 4%, 5%, 8%,
10% and 15% w/v). For example, 5% P4-AM was prepared by dissolving 50 mg P4-AM
in 1 ml potassium mono-di phosphate buffer (pH 8.0). 5% wlv P4-SG was prepared
by
dissolving 50 mg of P4-SG in milliQ de-ionized water. This solution was stored
on ice
until use. The networks were created following addition of a solution of P4-AM
with P4-
SG. For example, Formulation A contained 2% wlv solids. Formulation A cross-
linked
into a viscous branched polymer, Formulations B-G included equimolar amounts
of the
same components as in Formulation A, but at higher concentrations (B, 3% w/v
total
polymer; C, 4%; D, 5%; E, 8%, F, 10% and G, 15%). Formulations B-G cross-
linked
into tissue-conforming hydrogels i~ situ post-injection into muscle.
Incorporation of Plasmid DNA into the Network, and Effect on Gel Time
DNA was added to the solution containing P4-SG. 11.1 ~.1 of a 9mg/ml stock
solution of the nucleic acid (e.g., plasmid DNA or oligonucleotide) was added
to 38.8 p,1
of 6.4% solution of P4-SG, to obtain a 5% P4-SG solution containing 100 ~g
nucleic acid
in 100 ~,1. SO ~,1 of this P4-SG/DNA solution was then added to 50 ~,1 of 5%
w/v P4-AM
to formulate the desired gel of final concentration (5% total PEGs). The gel
time of this
formulation was approximately the same as a formulation that did not contain
nucleic
acid (5-6 minutes at 25~C).
Incorporation of a Third non- Nucleic Acid Polymeric Reagent, and the Effect
on
Gel Time
A third non-reacting, non-nucleic acid polymeric component was added to the
formulation. Methoxy-PEG2I~-di-stearoyl-phosphatidylethanolamine (mPEG-DSPE,
Genzyme) was selected as a polymeric excipient and added to the solution of P4-
SG. To
obtain a 10:1 mass ratio of DNA to PEG-DSPE, in a formulation containing 100
p,g
nucleic acid in 100 ~,1 of gel, 1 ~,1 of a 10 mg/ml mPEG-DSPE solution (in
milliQ de-
ionized water) was added to 38.7 ~,l of 6.5% P4-SG and 11.1 ~,1 of a 9 mg/ml
nucleic acid
solution prior to the addition of P4-AM. The final concentration of the gel
was 5% w/v
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reacting polymers (P4-AM+P4-SG). GeI formation was noted after mixing of the
solution. Addition of mPEG-DSPE to the formulation did not alter the gel time
at 25~C.
Network Characterization by Gel Permeation Chromatography
Gel permeation chromatography of formulation A (2% w/v P4-SH/P4-AM) was
performed to compare the size of unreacted components with that of the network
to
demonstrate formation of a high molecular weight, branched molecule of
molecular
weight ~l million. FIG 2A shows Gel permeation chromatograms of network
formulation A (2% PEGS) and the individual PEG components (P4-SG and P4-A1VI)
Network Characterization: Determination of Kinetics of Branching and Gelation
The time post-mixing to achieve maximum equilibrium branching or gel
formation was determined by changes in shear viscosity measured by a DV-II
Brookefield viscometer. The kinetics of hydrogel formation of formulations B-D
were
measured. The "onset of gelation", characterized by rapid increases in
solution viscosity,
indicated the gel time. Data shown in FIG. 2B demonstrates gelation kinetics
for
formulations A (2% w/v P4-AM/P4 SG), B (3% w/v P4-AM/P4-SG), C (4% w/v P4-
AM/P4-SG), and D (5% w/v P4-AM/P4-SG) measured by viscometry. As demonstrated
in FIG 2C, it is apparent that gels with a higher concentration of reacting
polymers gelled
faster than gels with a lower concentration of reacting polymer. As shown in
FIG 3, the
crosslinking reaction was accelerated at higher temperatures for P4-AM/P4-SG
formulations.
Network Characterization: Hardness or Softness of Gels
After gelation, the gels were removed from micro-centrifuge tubes and examined
for texture, and physical attributes. In analytical chemistry terminology, the
compression
moduli a (stresses /strain) of networks (dynes/cm~ were determined to
characterize
crosslink densities, or "mesh size." As demonstrated in the table in FIG 6, a
tightly
crosslinked hydrogel formed from higher concentrations has a higher
compression
modulus than a loosely connected network formed when mixing components with
lower
concentrations, and is therefore characterized as "harder" (e.g., 5% w/v P4-
AM/P4-SG
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gels were found to be "hard" whereas 3% w/v P4-AM/P4-SG gels were "soft").
Crosslink densities can control the ability of a molecule to diffuse through
the network.
Compatibility of Plasmid DNA with Gel Forming Components
A compatibility experiment was performed to ensure that pre-mixing components
1 and 2 did not decrease the integrity (e.g., supercoiling) of plasmid DNA.
Plasmid DNA
I O (pDNA) (10 ~,g/mI) was mixed with either of the reacting polymers (2% w/v
P4-AM or
P4-SG, 5% w/v P4-AM or P4-SG) and incubated at room temperature for 30
minutes.
FIG 4 demonstrates percent supercoiling of the pDNA as subsequently determined
by
agarose gel electrophoresis. DNA supercoiling was found to not be affected by
either of
the non-gelled components. In another experiment, plasmid DNA was incorporated
into
2% w/v and 3% w/v hydrogels, and then extracted into phosphate buffered saline
to test if
the supercoiling of the plasmid was compromised by the crosslinking reaction.
The
supercoiling of the network-extracted DNA was compared with control DNA that
had not
been incorporated into networks. No loss in DNA supercoiling was observed by
incorporation of plasmid in networks. As shown in FIGS.SA and SB, the data
demonstrates that DNA integrity was maintained in the presence of a cross-
linked
formulation.
Network Characterization: Iy~ ylt~o Equilibrium Swelling
Equilibrium swelling can be used to characterize hydrogels. This method, when
developed as a method of analysis, can be utilized effectively to determine
reproducibility of a formulation. Networks containing plasxnid DNA (with,
without
mPEG-DSPE) were prepared as described above except that the mixing was
performed in
a 96 well plate. Samples were incubated at 37°C for 1 hour to allow
complete gelation.
The gels were removed from the wells and placed into scintillation vials. The
vials were
weighed, and 5 ml of Dulbecco's phosphate-buffered saline (PBS) was added. The
vials
were incubated overnight at 37°C with gentle shaking. The buffer was
then aspirated out
of the vial and the gels were re-weighed. Equilibrium swelling was calculated
as
percentage increase in gel-weight using the following formula: % Swelling =
((Final
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Welght of Gel) 37~C~ 24 hrs (~tlal weight of Gel) 37oC 24 hrs ) (I~tial Welght
of Gel) 37°C, 24
hrs x 100.
Measurements were performed on formulations prepared with different
concentrations of the two reacting polymers, P4-AM and P4-SG, at different
intervals
following stock solution preparation. The graph in FIG SB demonstrates that
percent
swelling was unaffected for gels prepared at different intervals following
solution
preparation. The data in FIG SB demonstrate that it is evident that swelling
increases
with polymer concentration. Percent swelling is unaffected by the addition of
nucleic
acid (e.g., plasmid DNA at lmg/ml final concentration), 'or by the addition of
components
such as mPEG-DSPE. Thus, neither of these components is reactive with the gel
components.
Network Characterization: Izz-vitro Release of Plasmid DNA from P4-AM/P4-SG
Gels
In-vitro release of DNA from hydrogels B-D was measured by incubation of
plasmid-containing gels in phosphate buffered saline at 37°C (200 ~,1
hydrogel containing
200 ~g of plasmid in a scintillation vial was incubated in 2 ml of PBS). At
defined time
points, the supernatant was removed and transferred to a new tube. An
additional 2 ml of
PBS was then added to each vial and the samples were returned to the
incubator.
Percent DNA release from hydrogels was quantified using a DNA-NPR~ (Tosoh-
Biosep Inc.) anion exchange column using a gradient elution (HPLC Method:
Buffer A:
0.56M sodium chloride in 50 mM Tris, pH 9.0; Buffer B: 1.2M sodium chloride in
50
mM Tris, pH 9.0; 0-30% Buffer B in 15 minute gradient elution). A standard
curve was
constructed with control unformulated DNA diluted in PBS at various
concentrations and
analyzed by HPLC. Relaxed and supercoiled plasmid peaks were identified in
comparison with the retention time of the standards. FIG 7A and 7B demonstrate
iyz vitf°o
release data. FIG 7A shows a representative HPLC trace from DNA released from
formulation C (4% w/v P4-AM/P4-SG); the second peak in the triplet set of
peaks is
supercoiled DNA. FIG 7B shows cumulative release data from formulations B (3%
w/v
P4-AM/P4-SG), C (4% w/v P4-AM/P4-SG), and D (5% w/v P4-AM/P4-SG). The data
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indicate that plasmid can be released from the gels and that release is faster
with gels
containing a lower percentage of P4-AM and P4-SG.
Network Attributes: PEG-PEG Networks Protect Plasmid DNA from Serum
Endonucleases
100 p1 of cross-linked formulations (A=2% w/v P4-SG/P4-AM, B=3% w/v P4-
SG/P4-AM) containing 30 pg of ~3-gal DNA, were incubated with shaking at
37°C in 100
~1 of a solution containing fresh BALB/c mouse serum in dilution ratios 1:40
to 1:80 for
30 minutes. Controls were incubated in serum-free buffer.
Endonuclease-based digestion of unformulated DNA and DNA in network
formulations was compared by analysis of the plasmids on agarose gels.
I S FIG 8 demonstrates that both network formulations protected the plasmid
DNA
from serum endonucleases (lanes 5-6, 8-9), whereas unformulated DNA showed a
loss of
supercoiling after 30 minutes of incubation in both serum dilutions (lanes 4
and 7).
Network Characterization: Inj ectability
The injectability of the formulations was determined in the following manner:
P4-AM and P4-SG were loaded into separate 0.3 ml syringes, which were then
joined vii a syringe connector. Solutions of the components were mixed
rapidly, and
then retrieved into a single syringe. The mixed formulation was extruded
through a 26g
needle at various time points post-mixing. The time within which a formulation
could be
injected was recorded in minutes. FIG 6 demonstrates that formulations at
higher
concentrations gelled faster, lowering the time interval within which
injection could
occur.
Network Attributes: Iy~ yivo In Situ Gel Formation in Muscle Tissue
To determine if the network formulations could be injected into the muscle of
an
animal and would form networks iy~ situ ih vivo~ Evans Blue dye was added to
formulations made up of 2%, 3%, 4%, 5%, 8%, or 10% w/v of total PEGs (P4-AM/P4-
SG). The formulations were injected into the muscle tissue of mice.
Mice were sacrificed 60 minutes following injection, and the injected muscles
were removed and examined for visible gels.
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All formulations were injectable. Formulations containing 3%, 4%, 5%, 8%, and
10% w/v total PEGS (P4-AM+P4-SG) each formed transparent visible gels in the
muscle
tissue. The 2% w/v PEG formulation diffused throughout the muscle tissue as a
viscous
liquid.
Network Characterization: IjZ_vivo biodegradation
To study the biodegradation of the networks, 100 ~l of formulation B (3% P4-
SG/P4-AM) was inj ected per mouse, 50 p1 per anterior tibialis muscle, two
mice/group.
Network-containing muscle tissue was excised at predetermined time points and
the
muscle was digested using the tissue digestion method described below.
Materials for Enzyme Digestion Method
Cysteine-HCl (99.9% purified, Sigma)
EDTA (Sigma)
50% Papain solution, 99.9% purified (Sigma)
Collagenase (98% purified, Sigma)
Calcium Chloride, 10% w/w in deionized water.
Enzyme Digestion Method
0.06068 of cysteine-HCl and 2.9158 EDTA were weighed and added to a 100 ml
volumetric flask, then filled to the 100 ml mark. The pH of the solution was
adjusted to
6.25. The solution was bubbled with an inert gas such as argon to remove
oxygen, and
then stored at -20~C until use.
5 ml of the 50% papain solution (Sigma) was pipetted in a 10 ml volumetric
flask,
then filled to the 10 ml mark to 10 ml with the cysteine/EDTA buffer. The
solution was
stored at -20~C until use.
Collagenase (1.5 mg/ml) was prepared by weighing out 1.5 mg of collagenase and
resuspending in 1 ml of cysteine/EDTA buffer.
1125 ~,1 of the papain solution was added to a 15 ml centrifuge tube
containing the
tissue explant (weight of the explant should be between 30 and 600mgs). 1125
~,l of the
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collagenase solution and 750 ~l of 10% calcium chloride was added to the
centrifuge
tube and mixed. The centrifuge tube containing the explant and the digestion
cocktail
was equilibrated for 8-12 hours in a water bath maintained at 37~C. This step
resulted in
the digestion of the tissue. After digestion, the pH of the digested
dispersion was
adjusted to 11.5 with 30 p1 of 50% NaOH, and then the tube was placed in the
37~C bath
for an additional 8-12 hours. This step resulted in the digestion of the
crosslinked
network to the corresponding tetrameric PEG components. The pH of the solution
was
then adjusted back to 9 with aqueous HCI. The tissue debris was centrifuged
for 0.5
hour, and then filtered through a 0.22 ~m filter to prepare it for GPC
analysis of PEG.
Percent PEG remaining at the tissue site over time was analyzed by gel
permeation chromatography (Tosoh-Biosep TSK G3000PWXL column; mobile phase:
mM sodium monobasic phosphate buffer, pH 7.4).
The rate of i~_vivo bioabsorption of these polyethylene glycol-based networks
was determined by quantification of total PEGS remaining at the injected
muscle site over
time. The results demonstrate that ~40% of total injected PEGS for formulation
B had
20 cleared from the site 33 days post-injection, and that 60-80% of the total
injected PEG
polymer was lost approximately 90 days post injection. The study demonstrated
that the
network delivery systems can be usedin i~ vivo applications.
Example 2: Polyethylene oxide)-Poly(amidoamine) Networks yia formation
of amide linkages
Materials
Poly(amidoamine), (PAMAM), Generation 0 (GO), 4 amine groups (Dendritech)
Poly(amidoamine), (PAMAM), Generation 1 (G1), 8 amine groups (Dendritech)
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
Formulation
10% P4-SG (100 mg in 1 ml) was prepared in milliQ water arid stored on ice.
Equimolar concentrations of poly(amidoamine) GO (10 mM or 0.5%) and Gl (lOmM
or
0.5%) solutions were prepared by diluting the respective stock solutions in
phosphate
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buffer (pH 8.0). The P4-SG solution, when mixed in equal volumes with either
of the GO
or G1 solutions, formed a transparent soft gel. Gels with different
crosslinking density
could be formed by varying the concentrations of P4-SG and poly(amidoamine) GO
or
Gl . Different compositions (2%, 5%, 10%) containing PEG-DSPE and nucleic acid
were
also formulated, and all were found to be inj ectable.
Network Characterization: Determination ofly~ Ylt~o Equilibrium Swelling
To characterize the PAMAM/P4-SG hydrogels, equilibrium swelling studies were
performed. FIG 9 demonstrates that the amount of swelling is much lower for
these gels
than for the PEG hydrogels, and the addition of lipid was found to decrease
the swelling.
Example 3: Polyethylene oxide)-poly(ethyleneimine) networks yia formation
of amide linkages
Materials
Polyethyleneimine (PEI), 25 kD (Aldrich, Milwaukee, WI, USA)
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
Formulation Method
Ih situ crosslinking gels were formulated using poly(ethylenimine) and P4-SG.
A
10% w/v solution of P4-SG was prepared in milliQ water. 100 mM of PEI (0.15 %
w/v)
was prepared in phosphate buffer, pH 8Ø 100 ~,1 of this solution (10 times
molar excess)
was added to the P4-SG solution and quick gelation was observed (<1 minute).
Gelation time can be controlled by altering the PEI or P4-SG concentration,
and/or the pH of the solutions in which the individual polymers are
resuspended. For
example, a formulation containing 0.075% w/v PEI and 10% w/v P4-SG
reconstituted at
pH 8.0 has a gel time of 6 minutes at 25~C.
Example 4: Polyethylene oxide)-polyethylene oxide) networks (P4-SG/P4-
SIB via formation of thioester linkages
Materials
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
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Polyethylene oxide tetrasulfydryl (P4-SH) (SunBio Systems)
Method
100 ~,1 of a solution of P4-SG (3% w/v) in milliQ water was mixed with 100 p1
of
a solution of 3% w/v P4-SH in phosphate buffer, pH 8.0 in a 1.5 ml centrifuge
tube and
incubated at 37~C to form a 3% w/v hydrogel.
Similarly, gels of 4%, 10%, 20% and 30% w/v were also formed by mixing equal
volumes of equimolar solutions of the two network forming polymers. After 30
minutes,
the gels were retrieved and examined for their attributes.
The 3% w/v gels were found to be soft, whereas the 4-30% w/v gels were "hard"
and tightly crosslinked. Gel times for the 3%, 4%, and 10% w/v P4-SGlP4-SH
formulations were measured in a Brookefield viscometer at temperatures of 25~C
and
37~C. As shown in FIG. 10, gel times accelerated with increased polymer
concentrations.
Example 5: Modulation of Gene Expression In Murine Muscle Via
Modulation Of Network Density
Use of a plasmid that encodes a secreted protein permits serum sampling and
analysis for expressed protein without sacrificing the animal. For example,
plasmids
encoding secreted embryonic alkaline phosphatase gene, Factor VIII, Factor IX,
erythropoetin (EPO), endostatin, various cytokines, insulin, and bone
morphogenic
protein (BMP) have been used for this purpose. A plasmid encoding the human
secreted
embryonic alkaline phosphatase gene (pgWizTM SEAP, henceforth referred as
"SEAP")
was used to monitor systemic expression. SEAP, a secreted form of the membrane
bound
placental alkaline phosphatase, has a half life of from minutes to a few days
in serum. A
protein with a short half life is especially useful to reliably determine
expression kinetics.
Materials
pgWiz-SEAP, (Gene Therapy Systems Inc., San Diego, California, USA).
Polyethylene oxide-tetraamine (P4-AM) (SunBio Systems)
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
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mPEG-DSPE (Genzyme).
S-6 week old female C57BI6 mice (Jackson Laboratories, Bar Harbor, Maine,
USA)
5-6 week old DBA/2 and Rag2 mice (Taconic, Germantown, NY, USA).
Formulations
DNA was amplified and purified using a Qiagen Endo-freeTM kit (Qiagen Inc.,
Valencia, California, USA) or was purchased from Aldevron LLC (Fargo, North
Dakota,
USA).
All formulations were prepared by mixing of two solutions, one containing a
pre-
weighed amount of P4-AM dissolved in O.1M potassium phosphate buffer, pH 8.0,
and
the other containing an equimolar amount of P4-SG dissolved in cold deionized
water
containing SEAP plasmid DNA (100 p,g/100 ~,1 final volume of formulation) and
mPEG-
DSPE (10 ~g/100 ~,1 final volume of formulation).
Formulation A included 2% w/v P4-SG/P4-AM cross-linked into a viscous
branched polymer,
Formulations B-D (3, 4, and S% w/v P4-SG/P4-AM, respectively) included
equimolar amounts of the same components as A, but at higher concentrations.
These
formulations cross-linleed into tissue-conforming hydrogels iu situ post-
injection into
muscle.
The solutions were freshly prepared and injected into mouse muscle immediately
after mixing all formulation components.
Animal experiments
Mice were mildly anesthetized using isofluorane and injected with different
cross-
linked network formulations or with unformulated plasmid DNA (in saline)
bilaterally
into the anterior tibialis muscles. All animals were injected with 100 ~,g of
plasmid DNA
in an injection volume of 50 ~,l per muscle.
At different timepoints post-injection, mice were anesthetized and blood was
collected retro-orbitally. Serum was separated from red blood cells by
centrifugation and
stored at -80°C until assays were performed.
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SEAP assay
Levels of enzymatically active SEAP in mouse serum were measured using the
Tropix Phospha-Light~ luminometric assay kit (Applied Biosystems, Foster City,
California, USA). Assays were performed according to the manufacturer's
protocol
except that samples for the standard curve were prepared in normal mouse sera
(Stellar
Biosystems, Columbia, Maryland, USA) diluted 1:4.
All experimental serum samples were also diluted 1:4 in manufacturer-supplied
dilution buffer.
Luminescence measurements were performed using a Topcount~ plate reader
(Packard Instruments, Illinois) following 40 nvnutes of incubation in the
reaction buffer.
Serum SEAP levels at each timepoint were expressed in nanograms/ml using the
standard
curve generated from the positive control (purif ed human placental alkaline
phosphatase)
supplied with the assay kit. The data were further analyzed using a Thompson-
Tau
outlier analysis as described in Wheeler and Ganji, "Introduction to
Engineering
Experimentation," Prentice Hall, pp. 142-145 (1996) and plotted as averages
and standard
deviations.
Results
Administration of each of the network formulations resulted in detectable
levels
of serum SEAP for extended periods of time. FIG 11A shows that all networks
with
higher crosslink densities (i.e., formulations C and D) produced significant
serum levels
of SEAP expression compared to lightly crosslinked networks (i.e.,
formulations A and
B). To evaluate the long-term expression of DNA released from the network
formulations, percent positive animals (as measured by animals expressing more
than 300
pg/n~l of serum SEAP, a level that is 2-3 fold higher than background serum
SEAP levels
in saline injected mice) were plotted for each formulation FIG. 11B
demonstrates that
DNA delivery from the networks resulted in long-term expression of the encoded
protein
in serum, whereas protein levels in animals injected with unformulated DNA
dropped
precipitously after 3-4 weeks.
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One hypothesis for transient expression of proteins following intramuscular
injections of plasmids is antibody-directed complement-mediated cytotoxicity
(ADCC).
To evaluate if the sustained protein expression kinetics observed in
immunocompetent
animals was apparent in complement deficient mice incapable of mounting ADCC,
DBA/2J mice, deficient in a component of complement, were injected with
unformulated
DNA or DNA in network formulations. FIG 12A show that in complement-deficient
animals, network injection produced more sustained expression of SEAP compared
to
that produced by unformulated DNA.
To fiuther evaluate the effect of long-term protein expression in
immunodeficient
animals,1ZAG2 knock out mice, incapable of V(D)J recombination, and thus
lacking
mature B and T cells, were administered pSEAP plasmid as unformulated DNA or
in
network formulations. FIG 12B shows that serum SEAP levels from animals
injected
with network associated DNA were sustained longer than those from the groups
injected
with unformulated DNA.
Example 6: Delivery of nucleic acid in P4-AM/P4-SG networks followed by
electroporation enhances gene expression
Materials
Polyethylene oxide-tetraamine (P4-AM) (SunBio Systems)
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
mPEG-DSPE (Genzyme)
SEAP plasmid DNA (Gene Therapy Systems)
5-6 weelc old female C57BI6 mice (Jackson Laboratories)
Formulations
3% w/v P4-AM/P4-SG was formulated with mPEG-DSPE (10~,g/100p,1) and
(100~,g/100~1) SEAP DNA. The gel was identified as a GT20 gel. GT20 denotes a
gel
time of 20 minutes post reconstitution with buffer at pIi 8 as measured by
viscometry at
25~C.
Method
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Mice were mildly anesthetized using isofluorane and injected with different
crosslinked network formulations or with unformulated plasmid DNA (in saline)
bilaterally into the anterior tibialis muscles. All animals were injected with
100 pg of
plasmid DNA in an injection volume of 50 p,1 per muscle.
The mouse muscles were electroporated immediately post-injection of the
formulations with 200 V/cm, 8 pulses, 20 ms pulse width at 1 second intervals
(Genetronics electroporator, ECM 830; BTX Inc., San Diego, California, USA).
Serum collection, SEAP assays, and data analysis using Thompson-Tau Outlier
analysis were performed as in example 5.
The data shown in FIG. 13 demonstrates enhancement of SEAP expression in
network formulation by electroporation.
Example 7: Addition of Excipients To P4-AM/P4-SG Networks Enhances
Gene Expression
Materials
Polyethylene oxide-tetraamine (P4-AM) (SunBio Systems)
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
mPEG-DSPE (Genzyme)
SEAP plasmid DNA (Gene Therapy Systems)
5-6 week old female C57B16 mice (Jackson Laboratories)
Formulations
3 % w/v P4-AM/P4-SG was formulated with mPEG-DSPE (10 p,g/100 ~,1) and
(100 p,g/100 p1) SEAP DNA. The gel was also identified as a GT20 gel. Various
excipients were added to the DNA-containing P4-SG solution, before mixing with
the
P4-AM solution. The final concentrations of these excipients in the
formulation were:
sodium lauryl sulfate (SDS, 0.1% w/v)(Sigma), pluronic L62 (0.1% w/v)(BASF)
Magainin I (0.025% w/v) (Sigma), and poly(amidoamine) (PAMAM; Dentritech) GO
(0.15% w/v). More specifically, sodium lauryl sulfate is classified as a
anionic lipid,
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pluronic L62 as a non-ionic surfactant, Magainin I as a cationic peptide, and
PAMAM GO
as a cationic 4-armed polymer.
Method
Mice were mildly anesthetized using isofluorane and injected with different
cross-
linked network formulations or with unformulated plasmid DNA (in saline)
bilaterally
into the anterior tibialis muscles. All animals were injected with 100 ~,g of
plasmid DNA
in an injection volume of 50 ~,1 per muscle (n=8 per group).
Serum collection by retro-orbital bleeding, SEAP assays, and~data analysis
using
Thompson-Tau Outlier analysis were performed as in example 5.
FIGS 14A and 14B demonstrate that SEAP expression was found to be enhanced
by the addition of these excipients to the network formulations.
Example 8: Gene Expression In Mouse Muscle Induced By P4-SG/P4-SH
Networks
Materials
Polyethylene oxide)-tetrasulfydryl (P4-SH) (SunBio Systems)
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
mPEG-DSPE (Genzyme)
SEAP plasmid DNA (Gene Therapy Systems)
5-6 week old female C57B16 mice (Jackson Laboratories)
Formulations
DNA was amplified and purified using a Qiagen Endo-free~ kit (Qiagen Tnc.,
Valencia, California) or was purchased from Aldevron LLC (Fargo, ND).
All formulations were prepared by mixing of two solutions, one containing a
pre-
weighed amount of P4-SH dissolved in O.1M potassium phosphate buffer, pH 8.0,
and
the other containing an equimolar amount of P4-SG dissolved in cold deionized
water
containing SEAP plasmid DNA (100~g/100~,1 final volume of formulation) and
mPEG-
DSPE (10~,g/100~1 final volume of formulation).
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Two formulations were tested: Formulation A: 3.5% w/v of each P4-SH/P4-SG
gelled after 20 minutes at 25°C; and Formulation B: 5% w/v of each P4-
SH/P4-SG gelled
after 10 minutes at 25°C. The solutions were freshly prepared and
injected into mouse
muscle immediately after mixing all of the formulation components.
Animal experiments
Mice were mildly anesthetized using isofluorane and injected with different
cross-
linked network formulations or with unformulated plasmid DNA (in saline)
bilaterally
into the anterior tibialis muscles. All animals were injected with 100 ~,g of
plasmid DNA
in an injection volume of 50 ~,l per muscle. There were 8 animals per group.
At day 7 post-injection, mice were anesthetized blood was collected, serum
1 S prepared and analyzed as in Example S. As shown in FIG 15, formulations A
and B both
induced high levels of gene expression in mice.
Example 9: Network-Mediated (P4AM-P4-SG) Gene Expression in Mouse
Mucosa
Materials
pgWiz-SEAP, (Gene Therapy Systems Inc., San Diego, California).
Polyethylene oxide)-tetraamine (P4-AM) (SunBio Systems)
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
mPEG-DSPE (Genzyme)
5-6 week old female C57B16 mice (Jackson Laboratories)
Methods
DNA was amplified and purified using a Qiagen Endo-free~ kit (Qiagen Inc.,
Valencia, California, USA) or was purchased from Aldevron LLC (Fargo, ND,
USA).
All formulations were prepared by mixing of two solutions, one containing a
pre-
weighed amount of P4-AM dissolved in O.1M potassium phosphate buffer, pH 8.0,
and
the other containing an equimolar amount of P4-SG dissolved in cold deionized
water
containing SEAP plasmid DNA (100 ~g/100 ~,1 final volume of formulation) and
mPEG-
DSPE (10 ~,g/100 p1 final volume of formulation).
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GTS: 5% w/v of each of P4-AM and P4-SG gelled after 5 minutes at 25~C. The
solutions were freshly prepared and inj ected into mouse rectum immediately
post mixing,
of all formulation components.
Animal experiments
Mice were mildly anesthetized using isofluorane and injected with different
crosslinked network formulations or with unformulated plasmid DNA (in saline)
info the
rectum 3.5 cm from the anus. All animals were injected with 100 ~g of plasmid
DNA in
an injection volume of 50 ~,1. There were 5 animals per group.
At day 8 post-injection, mice were anesthetized blood was collected, serum
prepared and analyzed as in example 5. Animals receiving unformulated DNA did
not
show SEAP expression. GTS formulations induced significant levels of gene
expression
in 3 of 5 mice.
Example 10: Demonstration Of Immune Responses To DNA encoded
Antigen Following IM Injections with Plasmid in P4-AM/P4-SG networks
Materials
Polyethylene oxide)-tetraamine (P4-AM) (SunBio Systems)
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
mPEG-DSPE (Genzyme)
The synthetic peptide, TPHPARIGL, representing the naturally processed H-2 Ld
restricted T cell epitope spanning amino acids 876-884 of ~3-gal and
IPQSLDSWWTSL,
the H-2 Ld epitope corresponding to residues S28-39 of hepatitis B surface Ag
(HBsAg),
were synthesized by Multiple Peptide Systems (San Diego, CA) to a purity of
>90% as
assessed by reverse phase high-pressure liquid chromatography (RP-HPLC). The
identity of each peptide was confirmed by mass spectrometry.
pCMV/~3-gal encoding gscherichia coli (3-gal driven by the human CMV
intermediate early promoter was used as the reporter gene for all
immunizations.
BALBIc mice, 6-10 wk of age
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CT26.WT and CT26.CL2S cell lines. CT26.WT is a clone of CT26, a BALB/c
(H-2d) undifferentiated colon adenocarcinoma. CT26.CL2S is a CT26.WT clone
stably
transfected with the lacZ gene. Cell lines were maintained in RPMI 1640, 10%
heat-
inactivated fetal calf serum (FCS; Life Technologies, Grand Island, NY), 2mM L-
glutamine, 100 pg/ml streptomycin, and 100 Ulml penicillin (Life Technologies,
Grand
Island, N~. CT26.CL2S was maintained in the presence of 400 p,g/ml 6418
sulfate
(Life Technologies, Grand Island, NIA.
Formulations
All formulations were prepared by mixing of two solutions, one containing a
pre-
weighed amount of P4-AM dissolved in O.1M potassium phosphate buffer, pH 8.0,
and
1 S the other containing a pre-weighed amount of P4-SG dissolved in cold
deionized water
containing ~3-gal DNA (100~,g/100p,1 of formulation) and mPEG-DSPE
(lOpg/100~,1 of
formulation). Formulation A included 2 % w/v P4-AM/P4-SG and created a viscous
branched polymeric network post-mixing of the components. Formulation B
included
3% w/v P4-AM/P4-SG and formed a hydrogel post-mixing. The solutions were
freshly
prepared at room temperature, mixed and injected immediately.
Physico-chemical Characterization of the formulations
The molecular weight and size distribution profile of formulation A was
determined to be one million by aqueous gel permeation chromatography using a
TSK
Gel Mixed Bed column with 0.02M phosphate buffer, pH 7.5, as the mobile phase.
The
2S network had a fluid viscosity of ~S cp, as measured by Brookefield
rheometry. The gel
point of formulation B was ~l l minutes at 37°C as measured by
Brookefield rheometry.
Immunizations
Mice were mildly anesthetized using isoflurane and injected with different
crosslinked network formulations or saline bilaterally into the anterior
tibialis muscles.
All animals were injected a single time with 30 ~,g of plasmid DNA in an
injection
volume of SO ~,l per muscle. In a separate experiment, dissection of the
muscle site
approximately an hour post injection of formulation B demonstrated presence of
a
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hydrogel conformed to tissue. Examination of the muscle site an hour post-
injection of
formulation A demonstrated formation of a thick, viscous gelatinous material.
ELISA Assay
Sera was collected from mice by retro-orbital bleeding at 12 weeks post-
immunization. Titers of ~3-gal specific antibodies at 12 weeks were measured
by a
standard ELISA protocol. ~-gal titers were defined as the highest serum
dilution that
resulted in an absorbance (OD 405) value twice than that of non-immune sera at
that
dilution. FIG 16 demonstrates that administration of DNA in networks derived
from both
formulations stimulated robust ~-gal antibody responses measured 12 weeks post
injection. Sinular results were obtained in two separate experiments with
identical
formulation groups.Proliferative T Cell Responses
T cells from pooled (n=4) splenocytes of immunized or naive mice were purified
using T cell enrichment columns according to the manufacturer's instructions
(R&D
Systems, Minneapolis, MN) at 12 weeks post-immunization. T cell proliferation
assays
were performed by incubating purified T cells and syngeneic irradiated
splenocytes (2 x
105 each) in the presence of 30 ~,g/xnl of ~3-gal or chicken ovalbumin protein
at 37~C for
72 hrs. Cultures were pulsed with 1 ~Ci of tritiated thiamidine (3H-TdR) and
incubated
for 20 hours. Cells were then harvested and radioactivity measured on a beta
counter.
FIG 17 shows that delivery of DNA in both network formulations induced ~3-gal
specific
proliferative T cell responses. This type of response is usually associated
with a T helper
restricted T cell population. Similar results were obtained in two separate
experiments
with identical formulation groups.
Gamma-Interferon ELISpot
T cells from pooled (n=4) splenocytes of immunized or naive mice were purified
using T cell enrichment columns according to the manufacturer's instructions
(R&D
Systems, Minneapolis, Ml~ at 12 weeks post-immunization. Purified T cells (2 x
105)
were stimulated with 2 x 105 irradiated (3-gal or HBV peptide pulsed syngeneic
spleen
cells for 24 hrs. The MHC Class I restricted T cell response elicited by these
formulations was measured in a gamma-interferon (y-IFN) enzyme-linked
immunospot
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(ELISpot) assay according to the manufacturer's directions (R&D Systems, Cat#
EL485,
Minneapolis, MN, USA). Spots were enumerated electronically. FIG 18
demonstrates
that responses were detected at both the 12 week time points and were higher
in mice
given formulation A in comparison to those of mice receiving formulation B.
Tumor Protection Studies
IO Mice were challenged intravenously with 5x105 CT26.WT or CT25.CL25 cells
post immunization with formulated DNA or saline control. Mice were sacrificed
on day
13, lungs were isolated and stained with 0.2% X-gal solution after fixing with
0.25%
glutaradehydel0.01 % formalin in PBS. Tumor nodules could then be visualized
and
enumerated. The protective response to this tumor is dependent on the class I
restricted T
cell response. Examination of lungs harvested on day 13 after tumor
inoculation
indicated the presence of multiple pulmonary metastases in all mice challenged
with the
CT26.WT cell line. Mice immunized with network entrapped DNA and challenged
with
the CT26 ~3-gal expressing tumor (CT26.CL25) were protected from metastases.
As
demonstrated by the data in the table of FIG 19, all but one mouse had
completely clear
lungs.
Example 11: Preparation of A Lyophilized Formulation
A schematic of a method for formulating a "one vial" lyophilized product that
contains an excipient(s) such as a lipid, unreacted PEG-amine, unreacted PEG-
succinimidyl glutarate, and a nucleic acid is provided in FIG. 20. At pHs
greater than
7.0, the two PEG components mutually react to form a crosslinked network.
Therefore,
the pH of the solution containing the two PEG components was maintained below
this
threshold (e.g., the pH is maintained at 5.5 by the dissolution of the
components in
deionized water).
In this example, the reactivity of the two PEG components was also controlled
by
temperature. At 37°C, the gel-forming reaction proceeded at a faster
rate than it did at
4°C. Therefore, the reaction in this example was maintained at
approximately 0 to 4°C
(an ice water slurry).
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FIG 21 shows a schematic for characterization of gels at lower temperature.
After
the mixing of the components, vials containing the DNA were filled with the
solution and
then Lyophilized. The lyophile was reconstituted with phosphate buffered
saline, pH 8.0,
and gelation times (onset of gelation) were measured. A 3% w/v geI formed in
approximately 25 minutes at 25~C and did not vary from the gel time of a non-
lyophilized formulation.
Lyophilization was also performed by mixing solutions of the reactive polymers
(e.g., P4-SG and P4-SH), maintaining a pH of below 7, and lyophilizing in the
absence of
nucleic acid. In this instance, the nucleic acid was added to the formulation
upon
reconstitution. As shown in FIG 21, gel times for formulations prepared in
this way did
not vary by the Iyophilization procedure.
Solutions prepared from reconstituted vials were injected into mouse muscles
within 5-7 minutes after reconstitution using the same DNA dose, immunization
and
assay protocols as described in Example 10. Formulations injected were 2% w/v
P4-
SG/P4-AM and 3% w/v P4-SG/P4-AM. The MHC Class I restricted T cell response
elicited by these formulations was measured in a gamma-interferon (Y-IFN)
ELISPOT
assay according to the manufacturer's directions (R&D System, Minneapolis,
MN).
Spots were enumerated electronically. FIG 22 shows that responses for both
formulations were analyzed at 12 weeks post immunization. The results were
statistically
equivalent indicating that Lyophilization does not adversely affect the
ability of the
formulation to function in vivo.
Example 12: Generation of Networks (P4-AM/P4-SG) containing
oligonucleotides
Materials
Polyethylene oxide tetraamine (P4.-AM) (SunBio Systems)
Polyethylene oxide)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
Oligonucleotides with phosphorothioate or phosphodiester backbones (Oligos,
etc., Wilsonville, Oregon, USA)
4I
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Methods
~( ~Tof a solution of P4-SG (5% wlv in milliQ water) (100 fig) was mixed with
100 ~l of a 5% w/v P4-AM and oligophosphorothioate (1 ~,gl~,l) (in phosphate
buffer, pH
8.0) solution and incubated at 37°C. The onset of gelation was
determined to be
approximately 8 minutes at 37°C by Brookefield rheometry and the
formation of a soft
gel was confirmed.
Formulations at concentrations of 5 and 10% wlv PEGS (P4-AM and P4-SG) with
and without oligonucleotide were also prepared, and the formation of gels was
noted in
all cases.
In Vitf°o Release of Oligonucleotides
100 p,1 of a solution of P4-SG in milliQ water was mixed with 100 ~,l of a
solution
of P4-AM and 1 ~glp,l of oligophosphorothioate (in phosphate buffer, pH 8.0)
in a 1.5 ml
centrifuge tube and incubated at 37°C. After 1 hour, the gel was
retrieved and placed in a
new centrifuge tube with 1 ml of phosphate buffered saline, pH 7.4. The gels
were
incubated at 37°C. At each timepoint, 800 ~l of supernatant was
retrieved and transferred
to a new tube. To the tube containing the gel was added 800 p1 of fresh
buffer. The
supernatant was analyzed fox oligophosphorothioate content by anionic exchange
chromatography.
FIGS 23A and 23B show the results of iy~_vitro release assays that were
performed for 5%
and 10% hydrogels containing 1 p,g/ml of oligo.
example 13: P4-SH/P4-SG Networks Containing Oligonucleotides
Materials
Polyethylene oxide Tetrasulfydryl (P4-SH) (SunBio Systems)
Polyethylene oxide 3350)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
Oligonucleotides with phosphorothioate or phosphodiester backbones (Oligos,
etc.)
Formulations
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50 ~,1 of a solution of P4-SG (5 %w/v in mQ water) and oligophosphorothioate
(100 fig) was mixed with 50 ~,1 of 5 %w/v P4-AM (in phosphate buffer, pH 8.0)
solution
and incubated at 37°C. Additional formulations with 3%, 4%, 10%, 20%,
and 30% w/v
total PEGS containing oligophosphorothioate were also generated and the
formation of a
gel was noted in each case.
The kinetics of cross-linking of the hydrogels (3%, 4%,10%, 20%, 30%) was
measured by Brookefield Rheometry at 25°C. For each of these
formulations, the onset
of "gel" formation was characterized by the rapid increase in shear viscosity
that marked
the critical gel point, G~. Gel times for 20 and 30% w/v gels were less than 2
minutes at
25°C and 37°C. FIG 24A demonstrates that at 37°C, the
rate of gelation was faster. FIG
24B demonstrates that the gel time at higher pHs was faster and thus, the gel
time could
be modulated by variations in temperature and pH.
Ih Vitro Release of Oligonucleotides
100 ~,1 of a solution of P4-SG in milliQ water was mixed with 100 ~,l of a
solution
of P4-SH and l~,g/ml of oligophosphorothioate (in phosphate buffer, pH 8.0) in
a 1.5 ml
centrifuge tube and incubated at 37°C. After 1 hour, the gel was
retrieved and placed in a
new centrifuge tube with 1 ml of phosphate buffered saline, pH 7.4. The gels
were
incubated at 37°C. At each timepoint, 800 ~,l of supernatant was
retrieved and
transferred to a new tube. 800 ~,l of fresh buffer was added to the tube
containing the gel.
The supernatant was analyzed for oligophosphorothioate content by anionic
exchange
chromatography. Release assays were performed for 10, 20 and 30% hydrogels
containing 1 ~g/mI of oligo. FIG 25A and 25B show that in 14 days,the total %
ODN
released was ~98% for 10% gels, ~ 85% for 20% gels, and ~78% for 30% gels.
Example 14: PAMAM/P4-SG Networks Containing Oligonucleotides
Materials
Poly(amidoamine), Generation 0 (GO), 4 amine groups (Dendritech)
Polyethylene oxide 3350)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
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Oligonucleotides with phosphorothioate or phosphodiester backbones (Oligos,
etc.)
Network Formulations
50 ~,l of a solution of P4-SG (I O %w/v in mQ water) and oligophosphorothioate
(100 p,g) was mixed with 50 p,1 of 0.1 %w/v PAMAM, Generation 0 (in phosphate
buffer,
pH 8.0) solution and incubated at 37°C. The gel time was 4 minutes at
25°C.
Vitro Release of Oligonucleotides
100,1 of a solution of 10% w/v P4-SG in milliQ water was mixed with 100 ~,l of
a solution of 0.5% w/v PAMAM and 1 ~g/p,l of oligophosphorothioate (in
phosphate
buffer, pH 8.0) in a 1.5 ml centrifuge tube and incubated at 37°C.
After 1 hour, the gel
was retrieved and placed in a new centrifuge tube with 1 ml of phosphate
buffered saline,
pH 7.4. The gels were incubated at 37°C. At each time point, 800 p,1 of
supernatant was
retrieved and transferred to a new tube. To the tube containing the gel, was
added 800 ~,1
of fresh buffer. The supernatant was analyzed for oligophosphorothioate
content by
anionic exchange chromatography. FIG 26 demonstrates that approximately ~18%
of the
oligophosphorothioate was released within 1 day and 98.5% was released within
5 days.
Example 15: Micronized Calcium Phosphate Oligonucleotide In P4-AM/P4-
SG Networks
Materials
CaCl2: 0.1 M solution in deionized water (Sodium- and potassium-free calcium
hloride must be used) (Sigma)
Polyethylene oxide Tetrasulfydryl (P4-SH) (SunBio Systems)
Polyethylene oxide 3350)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
Oligonucleotides with phosphorothioate or phosphodiester backbones (Oligos,
etc.)
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Formulations
~1 of 0.1 M CaCl2 was added dropwise to a 100 p1 solution of oligophosphoro-
thioate in deionized water (1 mg/ml), while stirring. A fine white precipitate
formed in
the tube.
The white precipitate was dialyzed by centrifugation/filtration using a 1.5 ml
10 Centricon Filtrion~ centrifuge tube.
The white precipitate was reconstituted in a 3% wlv solution of P4-AM.
50 ~1 of the P4-AM/OligoCaP dispersion was added to 50 ~,1 of a 3% w/v P4-SG
solution to form a 3% total PEGs formulation. Gel time of a 3% PEGS geI with
micronized CaP-ODN was ~10 minutes at 37~C, and 19 minutes at 25 ~C.
The gel was characterized as a "hard" gel.
Example 16: Networks Containing Microparticles in Hydrogel
Materials
Poly(lactide-co-glycolide) microparticles (12,000 Daltons) containing plasmid
DNA (Aldveron, LLC)
Polyethylene oxide 3350)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
Polyethylene oxide) tetrasulfydryl (P4-SH) (SunBio Systems)
Formulation
10, 50, and 100 mg batches of DNA-containing microparticles were added to 50
~1 solutions of 10% w/v P4-SH (A, B, C, respectively) made up in phosphate
buffer, pH
8Ø 50 p1 of a solution of 10% w/v P4-SG made up in DI water was added to
solutions
A, B and C to make formulations A, B and C. Gel times and gel characteristics
were
determined.
Network Characterization
Formulations A, B and C all gelled after between 2-3 minutes at room
temperature, demonstrating no inhibition of gelation by addition of
microparticles.
Hydrogels fabricated from formulation C were found to be hard and brittle.
Hydrogels
from A and B were hard, but pliable. This study demonstrates the feasibility
of
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incorporation of microparticles into hydrogels for the purpose of applying
drug delivery
devices to rounded tissues and surfaces. The hydrogel in this case would hold
the
microparticles "in place."
Example 17: Chitosan/P4-SG Networks (CI3/P4-SG)
Materials
Chitosan, glutamate salt (Pronova)(CH), MW ~l million
Polyethylene oxide 3350)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
Plasmid DNA, SEAP (Gene Therapy Systems)
Formulation
A solution containing 0.05% chitosan glutamate (CH) was prepared in phosphate
buffer, pH 8Ø 501 of this solution was added to 501 of a solution containing
5% w/v
P4-SG and 1 p,g/~,1 DNA in DI water (CH/P4-SG).
Network Characterization: Gel Time, Hardness/Softness
The formulation gelled instantaneously at 25~C, forming a hard gel. This
formulation demonstrates the feasibility of a proteolytically degradable
network (e.g., a
network degradable by lysozymes).
Example 18: Poly(lysine)/P4-SG Networks (PL/P4-SG)
Materials
Poly(lysine) hydrobromide, MW 150,000 (Sigma)
Polyethylene oxide 3350)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
Plasmid DNA, SEAP (Gene Therapy Systems)
Formulation
A solution containing 1.0% w/v poly(lysine) hydrobromide (PL) was prepared in
phosphate buffer, pH 8Ø 501 of tlus solution was added to 50,1 of a solution
containing 5% wlv P4-SG and 1 ~g/~,1 DNA in DI water.
Network Characterization: Gel Time, Hardness/Softness
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The formulation gelled in between 2 and 3 minutes, and formed a semi-hard gel.
This formulation is another variation of a network formulation that can be
used for
nucleic acid delivery.
Example 19: (PEO-PPO-PEO-tetra-SI~/P4-SG Networks (PEO-PPO-
PEO/P4-SG)
Materials
Polyethylene oxide)-polypropylene oxide)-polyethylene oxide)-tetrasulfhydryl,
MW l OK Daltons (PEO-PPO-PEO-tetra-SH) (SunBio Systems)
Polyethylene oxide 3350)-tetrasuccinimidyl glutarate (P4-SG) (SunBio Systems)
Formulation
A solution containing 10% w/v PEO-PPO-PEO-tetra-SH was prepared in
phosphate buffer, pH 8Ø 50,1 of this solution was added to 50,1 of a
solution of 10%
w/v P4-SG and 1 ~g/~,1 DNA in DI water to form a 10% w/v gel.
Network Characterization: Gel Time, Hardness/Softness
The formulation gelled in 6-7 minutes, and formed a hard, oily gel. This
formulation is yet another variation of a network formulation that can be used
for nucleic
acid delivery.
Other Embodiments
It is to be understood that while the invention has been described in
conjunction
with the detailed description thereof, that the foregoing description is
intended to
illustrate and not limit the scope of the appended claims. Other aspects,
advantages, and
modifications are within the scope of the following claims.
What is claimed is:
47
