Delivery of AS-oligonucleotide microspheres to induce dendritic cell
tolerance for the treatment of autoimmune type 1 diabetes
United States Patent: 7,884,085
Issued: February 8, 2011
Inventors: Brown; Larry R.
(Newton, MA), Bisker-Leib; Vered (Woburn, MA), Scott; Terrence L.
(Winchester, MA), Lafreniere; Debra (Dighton, MA), Machen; Jennifer
(Export, PA), Giannoukakls; Nick (Coraopolis, PA)
International Inc. (Deerfield, IL),Baxter Healthcare S.A. (Glattpark (Opfikon),
CH), University of Pittsburgh-of the Commonwealth System of Higher
Education (Pittsburgh, PA)
Appl. No.: 11/127,360
Filed: May 12, 2005
George Washington University's Healthcare MBA
AS-oligonucleotides are delivered in
microsphere form in order to induce dendritic cell tolerance, particularly
in the non-obese-diabetic (NOD) mouse model. The microspheres incorporate
antisense (AS) oligonucleotides. A process includes using an antisense
approach to prevent an autoimmune diabetes condition in NOD mice in vivo
and in situ. The oligonucleotides are targeted to bind to primary
transcripts CD40, CD80, CD86 and their combinations.
Description of the
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to microsphere delivery of AS-oligonucleotides
in order to induce dendritic cell tolerance, particularly in the
non-obese-diabetic (NOD) mouse model. More particularly, the invention
relates to drug delivery technology by way of microspheres that are
fabricated using totally aqueous conditions, which microspheres
incorporate antisense (AS) oligonucleotides. These microspheres are used
for an antisense approach to prevent an autoimmune diabetes condition in
NOD mice in vivo and in situ.
2. Background of the Invention
Microparticles, microspheres, and microcapsules are solid or semi-solid
particles having a diameter of less than one millimeter, more preferably
less than 100 microns, which can be formed of a variety of materials,
including synthetic polymers, proteins, and polysaccharides. Microspheres
have been used in many different applications, primarily separations,
diagnostics, and drug delivery.
A number of different techniques can be used to make these microspheres
from synthetic polymers, natural polymers, proteins and polysaccharides,
including phase separation, solvent evaporation, emulsification, and spray
drying. Generally the polymers form the supporting structure of these
microspheres, and the drug of interest is incorporated into the polymer
structure. Exemplary polymers used for the formation of microspheres
include homopolymers and copolymers of lactic acid and glycolic acid (PLGA)
as described in U.S. Pat. No. 5,213,812 to Ruiz, U.S. Pat. No. 5,417,986
to Reid et al., U.S. Pat. No. 4,530,840 to Tice et al., U.S. Pat. No.
4,897,268 to Tice et al., U.S. Pat. No. 5,075,109 to Tice et al., U.S.
Pat. No. 5,102,872 to Singh et al., U.S. Pat. No. 5,384,133 to Boyes et
al., U.S. Pat. No. 5,360,610 to Tice et al., and European Patent
Application Publication Number 248,531 to Southern Research Institute;
block copolymers such as tetronic 908 and poloxamer 407 as described in
U.S. Pat. No. 4,904,479 to Illum; and polyphosphazenes as described in
U.S. Pat. No. 5,149,543 to Cohen et al. Microspheres produced using
polymers such as these exhibit a poor loading efficiency and are often
only able to incorporate a small percentage of the drug of interest into
the polymer structure. Therefore, substantial quantities of microspheres
often must be administered to achieve a therapeutic effect.
Spherical beads or particles have been commercially available as a tool
for biochemists for many years. For example, antibodies conjugated to
beads create relatively large particles specific for particular ligands.
The large antibody-coated particles are routinely used to crosslink
receptors on the surface of a cell for cellular activation, are bound to a
solid phase for immunoaffinity purification, and may be used to deliver a
therapeutic agent that is slowly released over time, using tissue or
tumor-specific antibodies conjugated to the particles to target the agent
to the desired site.
One disadvantage of the microparticles or beads currently available is
that they are difficult and expensive to produce. Microparticles produced
by these known methods have a wide particle size distribution, often lack
uniformity, and fail to exhibit long term release kinetics when the
concentration of active ingredients is high. Furthermore, the polymers
used in these known methods are dissolved in organic solvents in order to
form the microparticles. They must therefore be produced in special
facilities designed to handle organic solvents. These organic solvents
could denature proteins or peptides contained in the microparticles.
Residual organic solvents could be toxic when administered to humans or
In addition, the available microparticles are rarely of a size
sufficiently small to fit through the aperture of the size of needle
commonly used to administer therapeutics or to be useful for
administration by inhalation. For example, microparticles prepared using
polylactic glycolic acid (PLGA) are large and have a tendency to
aggregate. A size selection step, resulting in product loss, is necessary
to remove particles too large for injection. PLGA particles that are of a
suitable size for injection must be administered through a large gauge
needle to accommodate the large particle size, often causing discomfort
for the patient.
Generally, many currently available microparticles are activated to
release their contents in aqueous media and therefore must be lyophilized
to prevent premature release. In addition, particles such as those
prepared using the PLGA system exhibit release kinetics based on both
erosion and diffusion. In this type of system, an initial burst or rapid
release of drug is observed. This burst effect can result in unwanted side
effects in patients to whom the particles have been administered.
Microparticles prepared using lipids to encapsulate target drugs are
known. For example, lipids arranged in bilayer membranes surrounding
multiple aqueous compartments to form particles may be used to encapsulate
water soluble drugs for subsequent delivery as described in U.S. Pat. No.
5,422,120 to Sinil Kim. These particles are generally greater than 10
microns in size and are designed for intra articular, intrathecal,
subcutaneous and epidural administration. Alternatively, liposomes have
been used for intravenous delivery of small molecules. Liposomes are
spherical particles composed of a single or multiple phospholipid and
cholesterol bilayers. Liposomes are 30 microns or greater in size and may
carry a variety of water-soluble or lipid-soluble drugs. Liposome
technology has been hindered by problems including purity of lipid
components, possible toxicity, vesicle heterogeneity and stability,
excessive uptake and manufacturing or shelf-life difficulties.
An objective for the medical community is the delivery of nucleic acids to
the cells in an animal for diabetes treatment. For example, nucleic acids
can be delivered to cells in culture (in vitro) relatively efficiently,
but nucleases result in a high rate of nucleic acid degradation when
nucleic acid is delivered to animals (in vivo).
In addition to protecting nucleic acid from nuclease digestion, a nucleic
acid delivery vehicle must exhibit low toxicity, must be efficiently taken
up by cells and have a well-defined, readily manufactured formulation. As
shown in clinical trials, viral vectors for delivery can result in a
severely adverse, even fatal, immune response in vivo. In addition, this
method has the potential to have mutagenic effects in vivo. Delivery by
enclosing nucleic acid in lipid complexes of different formulations (such
as liposomes or cationic lipid complexes) has been generally ineffective
in vivo and can have toxic effects. Complexes of nucleic acids with
various polymers or with peptides have shown inconsistent results and the
toxicity of these formulations has not yet been resolved. Nucleic acids
also have been encapsulated in polymer matrices for delivery, but in these
cases the particles have a wide size range and the effectiveness for
therapeutic applications has not yet been demonstrated.
Therefore, there is a need for addressing nucleic acids delivery issues,
and there is an on-going need for development of microspheres and to new
methods for making microspheres. Details regarding microspheres are found
in U.S. Pat. No. 6,458,387 to Scott et al., U.S. Pat. Nos. 6,268,053,
6,090,925, 5,981,719 and 5,599,719 to Woiszwillo et al., and U.S. Pat. No.
5,578,709 to Woiszwillo. These and all references identified herein are
incorporated by reference hereinto.
SUMMARY OF THE INVENTION
In accordance with the present invention, DNA to be delivered to dendritic
cells is delivered as microspheres. It is believed that such a delivery
approach prevents access of the nucleases to the nucleic acids within the
microsphere. Microsphere delivery of AS-oligonucleotides is carried out in
order to induce dendritic cell tolerance, particularly in the NOD mouse
model. The microspheres are fabricated using aqueous conditions, which
microspheres incorporate antisense (AS) oligonucleotides. These
microspheres are used to inhibit gene expression and to prevent an
autoimmune diabetes condition in NOD mice in vivo and in situ.
In a preferred aspect of the invention, three AS-oligonucleotides targeted
to the CD40, CD80 and CD86 primary transcripts are synthesized, and an
aqueous solution of the oligonucleotide mixture is prepared and combined
with a polymer solution. After processing, microspheres containing the
oligonucleotides are provided, and these are delivered to the NOD mice.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As required, detailed embodiments of the present invention are disclosed
herein; however, it is to be understood that the disclosed embodiments are
merely exemplary of the invention, which may be embodied in various forms.
Therefore, specific details disclosed herein are not to be interpreted as
limiting, but merely as a basis for the claims and as a representative
basis for teaching one skilled in the art to variously employ the present
invention in virtually any appropriate manner.
The preferred embodiment prevents autoimmune insulin-dependent diabetes by
formulating and injecting antisense (AS)-oligonucleotide microspheres
described herein targeting the primary transcripts of CD40, CD80 and CD86.
These oligonucleotides are designed to induce immune tolerance in an
attempt to prevent destruction of the insulin producing beta cells in the
NOD mouse model. The events leading to the destruction of these beta cells
is illustrated in FIG. 1 (see Original Patent). This illustrates how Type
1 diabetes is manifested by the autoimmune destruction of the pancreatic
insulin-producing beta cells in the NOD mouse, as well as in humans. At
the time of clinical onset, humans have 10-20% residual beta cell mass.
Sparing of this residual mass can result in remaining insulin levels which
are adequate to regulate glucose levels. The microparticles of the
invention are provided to interfere with the autoimmune destruction of the
beta cells which is illustrated in FIG. 1.
It will be appreciated that dendritic cells (DC) can be activated to be
potent antigen presenting cells found in all tissues and which are highly
concentrated under the skin. These antigen presenting dendritic cells
function as triggers of the immune response through the activation of
T-cells, particularly in lymph nodes.
FIG. 2 (see Original Patent) is a drawing of a plasmid vector containing
the Beta-galactosidase gene that can be used to transfect NIH 3T3
fibroblast cells. In vitro evidence for the transfection of NIH 3T3
fibroblast cells with the plasmid DNA microspheres is shown in FIG. 3 by
the cells which stain blue in color in response to the addition of the
FIG. 4 (see Original Patent) illustrates the ability of microspheres to
protect DNA in solution. This is an agarose electrophoresis gel showing
nuclease protection imparted by microspheres of plasmid DNA produced
generally as noted herein. In the Plasmid samples 1, 2 and 3, naked
plasmid DNA was exposed to DNAse, with the smears indicating plasmid DNA
degradation at each of the three levels of DNAase application. In the
Particle 1 and Particle 2 samples, plasmid DNA microsphere formulations
were exposed to DNAase. The lack of smearing indicates the microsphere
formulations show shielding of the plasmid DNA from degradation. Particle
1 plasmid DNA samples show enhanced protection over Particle 2 plasmid DNA
FIG. 5 (see Original Patent) reports on Beta-Galactosidase activity of
four different plasmid DNA applications when transfected into cells. The
naked plasmid DNA application showed very low levels. Somewhat greater
levels are indicated for plasmid DNA cationic lipid complex application
using lipofectamine, a commercial cationic lipid, as the delivery vehicle.
Substantially greater activity is shown for two pDNA microspheres, with
Microspheres 1 corresponding to Particle 1 of FIG. 4, and Microspheres 2
corresponding to Particle 2 of FIG. 4.
In making the microspheres that are used for autoimmune treatment of
diabetes in mice, three AS-oligonucleotides are dissolved in aqueous
solution and combined with water soluble polymer(s) and a polycation. The
solution typically is incubated at about 60-70.degree. C., cooled to about
23.degree. C., and the excess polymer is removed. Microspheres are formed
which are believed to contain the three AS-oligonucleotides having the
following sequences, wherein an asterisk indicates thioation
-- see Original Patent.
More particularly, the nucleic acids typically comprise between about 30
and about 100 weight percent of the microspheres and have an average
particle size of not greater than about 50 microns. Typically, they are
prepared as follows. An aqueous solution of the oligonucleotide mixture is
prepared by combining aliquots from three oligonucleotide solutions, each
solution containing one of these three types.
A solution containing the three types of oligonucleotides is prepared. The
solutions preferably contain about 10 mg/ml oligonucleotide. These are
combined with aliquots of a 10 mg/ml stock solution of polycation solution
at volumetric ratios of polycation:oligonucleotide of from about 1:1 to
about 4:1. Polymer solutions of polyvinyl pyrrolidone and/or of
polyethylene glycol are prepared and combined with the other solutions.
Heating, cooling, centrifuging and washing multiple times provide an
aqueous suspension which typically is frozen and lyophilized to form a dry
powder of microspheres comprising oligonucleotide and polycation.
Microspheres according to the invention are a viable non-viral delivery
tool for plasmid DNA and antisense oligonucleotides and other nucleic
acids. They allow for in vitro delivery of Beta-Galactosidase plasmid DNA
in 3T3 fibroblast cells. The microspheres protect plasmid DNA from
nuclease activity. High levels of Beta-Galactosidase activity are
expressed following transfection with the microsphere formulations.
Microspheres containing the antisense oligonucleotides of interest
down-regulate surface cell antigens CD40, CD80 and CD86, known to be
critical in the activation of the autoimmune reaction that results in
destruction of insulin-producing beta cells of the pancreas. This can be
accomplished by subcutaneous injection to dendritic cells located under
the skin. NOD mice studies demonstrate effective prevention of the
autoimmune destruction of beta cells. The DNA and oligonucleotide
microspheres are effective transfection vehicles in vitro and in vivo.
Dendritic cells appear to take up the oligonucleotide microspheres and
suppress the expression of surface cell antigens CD40, CD80 and CD86. The
anitsense oligonucleotide microspheres effectively prevent diabetes
development in the NOD mouse.
The following Examples illustrate certain features and advantages of the
invention in order to further illustrate the invention. The Examples are
not to be considered limiting or otherwise restrictive of the invention.
Three AS-oligonucleotides targeted to the CD40, CD80 and CD86 primary
transcripts were synthesized by the DNA synthesis facility at University
of Pittsburgh (Pittsburgh, Pa.). The AS-oligonucleotides sequences are
-- see Original Patent.
An aqueous solution of the oligonucleotide mixture was prepared by
combining aliquots of three oligonucleotide solutions, each of which
contained one type of oligonucleotide, to form a 10 [mg/ml] solution of
the three types of oligonucleotides. 10 [mg/ml] poly-L-lysine.HBr in diH2O
(poly-L-lysine.HBr up to 50,000 by Bachem, King of Prussia, Pa.) was
prepared. Poly-L-lysine.HBr was added to the oligonucleotides solution at
a volumetric ratio of 1:1. The mixture was vortexed gently. A 25% polymer
solution containing 12.5% PVP (polyvinyl pyrrolidone, 40,000 Daltons,
Spectrum Chemicals, Gardena, Calif.) and 12.5% PEG (polyethylene glycol,
3,350 Daltons, Spectrum Chemicals, Gardena, Calif.) in 1M Sodium Acetate
(Spectrum, Gardena, Calif.) at pH=5.5 was made. The polymer solution was
added in a 2:1 volumetric ratio as follows: 750 .mu.l of AS-oligonucleotides,
0.75 ml of poly-L-lysine.HBr, 3.0 ml of PEG/PVP, and a total volume of
The batch was incubated for 30 minutes at 70.degree. C. and then cooled to
23.degree. C. Upon cooling, the solution became turbid and precipitation
occurred. The suspension was then centrifuged, and the excess PEG/PVP was
removed. The resulting pellet was washed by resuspending the pellet in
deionized water, followed by centrifugation and removal of the
supernatant. The washing process was repeated three times. The aqueous
suspension was frozen and lyophilized to form a dry powder of microspheres
comprising oligonucleotide and poly-L-lysine.
FIG. 6 (see Original Patent) presents a scanning electron micrograph (SEM)
of the 1:1 poly-L-lysine:oligonucleotide ratio material. Microspheres,
0.5-4 .mu.m in size, with an average particle size of approximately 2.5 .mu.m
were fabricated. Precipitation of an unknown material was also observed.
Additional studies by HPLC determined that the precipitation was comprised
of residual PEG/PVP, mostly PVP.
AS-oligonucleotides targeted to the CD40, CD80 and CD86 primary
transcripts were the AS-oligonucleotides sequences of Example 1. An
aqueous solution of the oligonucleotide mixture was prepared by combining
aliquots of the three oligonucleotide solutions, each of which contained
one type of oligonucleotide, to form a 10 [mg/ml] solution of the three
types of oligonucleotides. A solution of oligonucleotide mixture was
prepared. 5 [mg/ml] poly-L-ornithine.HBr in diH.sub.2O (poly-L-ornithine.HBr
11,900 (vis) by Sigma) was prepared. Poly-L-ornithine.HBr was added to the
oligonucleotides solution. The mixtures were vortexed gently. A 25%
polymer solution containing 12.5% PVP (40,000 Daltons, Spectrum Chemicals,
Gardena, Calif.) and 12.5% PEG (3,350 Daltons, Spectrum Chemicals,
Gardena, Calif.) in 0.1.M Sodium Acetate (Spectrum Chemicals, Gardena,
Calif.) at pH=5.5 was made. The polymer solutions were added. Incubation
and rinses followed as described in Example 1. 1.5 ml of the AS-oligonucleotides,
1.5 ml of the poly-L-ornithine.HBr, 3 ml of the PEG/PVP, and a total
volume of 6.0 ml was prepared.
FIG. 7 (see Original Patent) presents an SEM of this 1:1 poly-L-ornithine:oligonucleotide
ratio material. Microspheres, 0.2-8 .mu.m in size, with an average
particle size of approximately 2 .mu.m were fabricated. Precipitation of
an unknown material was also observed. Additional HPLC studies were able
to prove that this precipitation was comprised of residual PEG/PVP, mostly
In vivo studies were conducted using the NOD mouse model of Type 1
diabetes mellitus. Type 1 diabetes is manifested by the autoimmune
destruction of the pancreatic insulin-producing beta cells as illustrated
in FIG. 1. AS-oligonucleotides were used in three applications in an
attempt to interfere with the autoimmune destruction of beta cells. The
goal was to interfere with the dendritic cell function by targeting the
primary transcripts of CD40, CD80 and CD86, which encode dendritric cell
surface proteins required for T-cell activation. Dendritic cells with low
levels of CD40, CD80 and CD86 are known to promote suppressive immune cell
networks in vivo. These cascades can result in T-cell hyporesponsiveness
to beta cells in vivo.
In the first group of test animals, dendritic cells were propagated ex
vivo from bone marrow progenitors of NOD mice. Combinations of the three
AS-oligonucleotides targeting the primary transcripts of CD40, CD80 and
CD86 were added to the cells in tissue culture. After incubation, the AS-oligonucleotide
transfected dendritic cells were injected into syngenetic recipients of 5
to 8 weeks of age (not yet diabetic). This is a known ex-vivo delivery
In parallel, AS-oligonucleotide microspheres were injected directly into
other NOD mice of the same age. A single injection was carried out on each
thus-treated mouse. Another group of these NOD mice was not treated and
served as a control.
FIG. 8 (see Original Patent) shows that the control, untreated NOD mice
all developed diabetes by age 23 weeks. The ex vivo AS-oligonucleotide
transfected and re-infused dendritic cells group (AS-ODN DC) showed
delayed development of diabetes, with 20% remaining "Diabetes Free",
indicating glucose levels are maintained within a non-diabetic range. Of
the microspheres in vivo-injected NOD mice, 71% remained "Diabetes Free"
at 43 weeks.
Claim 1 of 23 Claims
1. A composition that comprises
microspheres comprising oligonucleotides for treatment of type 1 diabetes,
wherein said microspheres contain a first antisense sequence that targets
a primary transcript of CD40, a second antisense sequence that targets a
primary transcript of CD80, and a third antisense sequence that targets a
primary transcript of CD86, wherein each of said first, second and third
oligonucleotides reduces or suppresses in vivo expression of CD40, CD80
and CD86 respectively, and wherein said oligonucleotides comprise greater
than about 30 weight percent of the microspheres, based on the total
weight of the microspheres, said microspheres having an average particle
size of not greater than about 50 microns and at least 0.2 to 8 microns,
and said microspheres, when administered, treat type 1 diabetes.
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