U.S. patent application number 10/663265 was filed with the patent office on 2004-07-01 for polymeric gene delivery system.
Invention is credited to Boekelheide, Kim, Jong, Yong Shik, Mathiowitz, Edith.
Application Number | 20040126884 10/663265 |
Document ID | / |
Family ID | 22796022 |
Filed Date | 2004-07-01 |
United States Patent
Application |
20040126884 |
Kind Code |
A1 |
Mathiowitz, Edith ; et
al. |
July 1, 2004 |
Polymeric gene delivery system
Abstract
A means for obtaining efficient introduction of exogenous genes
into a patient, with long term expression of the gene, is
disclosed. The gene, under control of an appropriate promoter for
expression in a particular cell type, is encapsulated or dispersed
with a biocompatible, preferably biodegradable polymeric matrix,
where the gene is able to diffuse out of the matrix over an
extended period of time, for example, a period of three to twelve
months or longer. The matrix is preferably in the form of a
microparticle such as a microsphere (where the gene is dispersed
throughout a solid polymeric matrix) or microcapsule (gene is
stored in the core of a polymeric shell), a film, an implant, or a
coating on a device such as a stent. The size and composition of
the polymeric device is selected to result in favorable release
kinetics in tissue. The size is also selected according to the
method of delivery which is to be used, typically injection or
administration of a suspension by aerosol into the nasal and/or
pulmonary areas. The matrix composition can be selected to not only
have favorable degradation rates, but to be formed of a material
which is bioadhesive, to further increase the effectiveness of
transfer when administered to a mucosal surface.
Inventors: |
Mathiowitz, Edith;
(Brookline, MA) ; Jong, Yong Shik; (Seoul, KR)
; Boekelheide, Kim; (Wakefield, RI) |
Correspondence
Address: |
MINTZ, LEVIN, COHN, FERRIS, GLOVSKY
AND POPEO, P.C.
ONE FINANCIAL CENTER
BOSTON
MA
02111
US
|
Family ID: |
22796022 |
Appl. No.: |
10/663265 |
Filed: |
September 16, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10663265 |
Sep 16, 2003 |
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09815807 |
Mar 23, 2001 |
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6620617 |
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09815807 |
Mar 23, 2001 |
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08978522 |
Nov 25, 1997 |
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6262034 |
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08978522 |
Nov 25, 1997 |
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08467811 |
Jun 6, 1995 |
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08467811 |
Jun 6, 1995 |
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08213668 |
Mar 15, 1994 |
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Current U.S.
Class: |
435/455 |
Current CPC
Class: |
A61K 9/2027 20130101;
A61K 9/1647 20130101; A61K 48/00 20130101; A61K 9/1272
20130101 |
Class at
Publication: |
435/455 |
International
Class: |
C12N 015/85 |
Claims
1. A method of transfecting cells in vitro in culture medium
comprising contacting cells in a culture medium with a composition
for delivery of naked DNA, said composition comprising: (a) a
preparation of microparticles between 1 and 300 .mu.m in diameter,
each of which preparation of microparticles comprises a synthetic,
biocompatible, biodegradable polymeric matrix; and (b) an effective
amount of naked DNA dispersed within the preparation of
microparticles, wherein said amount of naked DNA is greater than 20
.mu.g, in which the DNA contains a gene operably linked to a
promoter, the nucleotide sequence of said gene being greater than
thirty nucleotides in length; wherein said DNA is released or
diffused from said matrix over a period of at least three
months.
2. A method of transfecting cells in vitro in culture medium
comprising contacting cells in a culture medium with a composition
for delivery of naked DNA, said composition comprising: (a) a
preparation of microparticles between 1 and 300 .mu.m in diameter,
each of which preparation of microparticles comprises a synthetic,
biocompatible, non-biodegradable polymeric matrix; and (b) an
effective amount of naked DNA dispersed within the preparation of
microparticles, wherein said amount of naked DNA is greater than 20
.mu.g, in which the DNA contains a gene operably linked to a
promoter, the nucleotide sequence of said gene being greater than
thirty nucleotides in length; wherein said DNA releases or diffuses
from said matrix over a period of at least three months.
Description
BACKGROUND OF THE INVENTION
[0001] The present invention is generally in the area of drug
delivery devices and is specifically in the area of polymeric drug
delivery devices.
[0002] Gene Therapy is generally defined as the introduction and
expression of an exogenous gene in an animal to supplement or
replace a defective or missing gene. Examples that have received a
great deal of recent attention include the genes missing in cystic
fibrosis and severe combined immunodeficiency. Although tremendous
progress has been made in the area of gene therapy, obtaining long
term expression of the desired proteins remains elusive.
[0003] In the majority of cases, a retroviral vector is used to
introduce the gene to be expressed into appropriate cells. Gene
transfer is most commonly achieved through a cell-mediated ex vivo
therapy in which cells from the blood or tissue are genetically
modified in the laboratory and subsequently returned to the
patient. The clinical studies by Steven Rosenberg, et al.,
"Immunotherapy of patients with metastatic melanoma using
tumor-infiltrating lymphocytes and IL-2", Preliminary report, New
England J. Med., 319 (1988) 1676-1680, using in vitro-activated LAK
and TIL for tumor destruction illustrates this approach. In other
cases, the vector carrying the gene to be expressed is introduced
into the patient, for example, by inhalation into the lungs in the
case of cystic fibrosis. Transfected cells have also been
implanted, alone or encapsulated within a protective membrane that
protects the cells from the inflammatory response of the body while
at the same time allowing the gene product to diffuse out of the
membrane. There have also been reports of the direct injection of
an exogenous gene in combination with an appropriate promoter, into
tissue, with some transient expression being noted.
[0004] Viral vectors have been widely used in gene transfer, due to
the relatively high efficiency of transfection and potential long
term effect through the actual integration into the host's genome.
However, there are still concerns about the risks involved in the
use of viruses. Activation of proto-oncogenes and reversion to
wild-type viruses from replication incompetent viruses are some
important potential hazards of viral delivery of genes.
[0005] Since the discovery that naked DNA is taken up by muscle
cells and transiently expressed in vivo, and subsequent reports, by
Wolff, Jon Aal, et, "Direct gene transfer into mouse muscle in
vivo," Science, 247, 1465-1468, 1990; and Wolff, Jon A, "Human
dystrophin expression in mdx mice after intramuscular injection of
DNA constructs," Nature, 352, 815-818, 1991, there has been
increasing interest in using non-viral vehicles for in vivo
transfections.
[0006] Plasmid DNA, which can function episomally, has been used
with liposome encapsulation, CaPO4 precipitation and
electroporation as an alternative to viral transfections. Recent
clinical trials with liposome encapsulated DNA in treating melanoma
illustrates this approach to gene therapy, as reported by Nabel, J.
G., et al., "Direct gene transfer with DNA-liposome complexes in
melanoma: Expression, biological activity and lack of toxicity in
humans", Proc. Nat. Acad. Sci. U.S.A., 90 (1993) 11307-11311. A
foreign gene coding for HLA-B was introduced into subcutaneous
sites of melanoma tumors. Expression of the new gene and the
absence of an anti-DNA host response was confirmed. Wolff, Jon A,
"Persistence of plasmid DNA and expression in rat brain cells in
vivo," Experimental Neurology, 115, 400-413, 1992, also reported
expression of plasmid DNA. Thus, direct gene transfer offers the
potential to introduce DNA encoding proteins to treat human
diseases.
[0007] The mechanisms for cellular uptake of exogenous DNA and
subsequent expression are not clear but gene transfer with naked
DNA is associated with several characteristics. Unlike in the case
of oligonucleotides, which are typically a maximum of twenty to
thirty nucleotides in length, genes encoding most molecules of
therapeutic interest are quite large, and therefore considerably
more difficult to introduce into cells other than through
retroviral vector, or in vitro, by chemical manipulation, so that
the efficiency of transfer is low. In most reported cases to date,
only transient expression of up to a few weeks or months has been
observed. Although low level expression and short term expression
are two important drawbacks with direct DNA transfer, transfections
with naked DNA have several advantages over viral transfers. Most
importantly, concerns related to the immunogenicity and
transforming capability of viruses are avoided. In addition, naked
DNA is easy to produce in large quantities, is inexpensive, and can
be injected at high concentration into localized tissue sites
allowing gene expression in situ without extensive ex vivo
therapy.
[0008] The following additional articles review the current state
of gene therapy and the problems associated therewith: Blau, Helen
M, "Muscling in on gene therapy," Nature, 364, 673-675, 1993;
Cohen, Jon, "Naked DNA points way to vaccines," Science, 259,
1691-1692, 1993; Dagani, Ron, "Gene therapy advance, anti-HIV
antibodies work inside cells," C&EN, 3-4, 1993; Felgner, Philip
L, "Lipofectamine reagent: A new, higher efficiency polycationic
liposome transfection reagent," Focus/Gibco, 15, 73-78, 1993; Liu,
Margaret A al, et, "Heterologous protection against influenza by
injection of DNA encoding a viral protein," Science, 259,
1745-1749, 1993; Marx, Jean, "A first step toward gene therapy for
hemophilia B," Science, 262, 29-30, 1993; Mulligan, Richard C, "The
basic science of gene therapy," Science, 260, 926-931, 1993;
Nicolau, Claudeal, et, "In vivo expression of rat insulin after
intravenous administration of the liposome-entrapped gene for rat
insulin I," Proc. Natl. Acad. Sci. USA, 80, 1068-1072, 1983;
Partridge, Terence A, "Muscle transfection made easy," Nature, 352,
757-758, 1991; Wilson, James M, "Vehicles for gene therapy,"
Nature, 365, 691-692, 1993; Wivel, et al., "Germ-line gene
modification and disease prevention: Some medical and ethical
perspectives," Science, 262, 533-538, 1993; and Woo, Savio L Cal,
et, "In vivo gene therapy of hemophilia B: sustained partial
correction in Factor IX-deficient dogs," Science, 262, 117-119,
1993.
[0009] Gene therapy is one of the most promising areas of research
today. It would therefore be extremely useful if one had an
efficient way to introduce genes into cells which yielded long term
expression.
[0010] It is therefore an object of the present invention to
provide a means for efficient transfer of exogenous genes to cells
in a patient.
[0011] It is a further object of the present invention to provide a
means for long term expression of exogenous genes in patients.
[0012] It is a further object of the present invention to provide a
means for increasing or decreasing the inflammatory response to
implanted polymeric devices.
[0013] It is a still further object of the present invention to
provide a method for immunization of individuals over a more
prolonged period of time than is achieved by a single or multiple
immunization protocol.
[0014] It is another object of the present invention to provide a
method for targeting of gene delivery either to tissue cells or to
inflammatory type cells.
SUMMARY OF THE INVENTION
[0015] A means for obtaining efficient introduction of exogenous
genes into a patient, with long term expression of the gene, is
disclosed. The gene, under control of an appropriate promoter for
expression in a particular cell type, is encapsulated or dispersed
with a biocompatible, preferably biodegradable polymeric matrix,
where the gene is able to diffuse out of the matrix over an
extended period of time, for example, a period of three to twelve
months or longer. The matrix is preferably in the form of a
microparticle such as a microsphere (where the gene is dispersed
throughout a solid polymeric matrix) or microcapsule (gene is
stored in the core of a polymeric shell), although other forms
including films, coatings, gels, implants, and stents can also be
used. The size and composition of the polymeric device is selected
to result in favorable release kinetics in tissue. The size is also
selected according to the method of delivery which is to be used,
typically injection into a tissue or administration of a suspension
by aerosol into the nasal and/or pulmonary areas. The matrix
composition can be selected to not only have favorable degradation
rates, but to be formed of a material which is bioadhesive, to
further increase the effectiveness of transfer when administered to
a mucosal surface, or select d not to degrade but to release by
diffusion over an extended period.
[0016] Examples demonstrate the effectiveness of the system in
animals.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Gene transfer is achieved using a polymeric delivery system
which releases entrapped genes, usually in combination with an
appropriate promoter for expression of the gene, into surrounding
tissue. Efficacy of transfer is achieved by: a) releasing the gene
for prolonged period of time, b) minimizing diffusion of the gene
out of the delivery system (due to its size) so that release is
predominantly degradation dependent, and c) improving the transient
time of expression and the low infection seen by direct gene
therapy. In case of non-erodible polymers, the device is formulated
so that the gene is released via diffusion. This is achieved by
creating porous systems or adding soluble bulking agents that
create pores as they leach out of the system.
[0018] The Polymeric Matrices
[0019] Selection of Polymer
[0020] Both non-biodegradable and biodegradable matrices can be
used for delivery of genes, although biodegradable matrices are
preferred. These may be natural or synthetic polymers, although
synthetic polymers are preferred due to the better characterization
of degradation and release profiles. The polymer is selected based
on the period over which release is desired, generally in the range
of at least three months to twelve months, although longer periods
may be desirable. In some cases linear release may b most useful,
although in others a pulse release or "bulk release" may provided
more effective results. The polymer may be in the form of a
hydrogel (typically in absorbing up to about 90% by weight of
water), and can optionally be crosslinked with multivalent ions or
polymers.
[0021] High molecular weight genes can be delivered partially by
diffusion but mainly by degradation of the polymeric system. In
this case, biodegradable polymers, bioerodible hydrogels, and
protein delivery systems are particularly preferred. Representative
synthetic polymers are: polyamides, polycarbonates, polyalkylenes,
polyalkylene glycols, polyalkylene oxides, polyalkylene
terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl
esters, polyvinyl halides, polyvinylpyrrolidone, polyglycolides,
polysiloxanes, polyurethanes and co-polymers thereof, alkyl
cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, polymers of acrylic and methacrylic
esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,
hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,
cellulose acetate, cellulose propionate, cellulose acetate
butyrate, cellulose acetate phthalate, carboxylethyl cellulose,
cellullose triacetate, cellulose sulphate sodium salt, poly(methyl
methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),
poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), poly(vinyl
acetate, poly vinyl chloride, polystyrene and
polyvinylpyrrolidone.
[0022] Examples of non-biodegradable polymers include ethylene
vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and
mixtures thereof.
[0023] Examples of biodegradable polymers include synthetic
polymers such as polymers of lactic acid and glycolic acid,
polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid),
poly(valeric acid), and poly(lactide-co-caprolactone), and natural
polymers such as alginate and other polysaccharides including
dextran and cellulose, collagen, chemical derivatives thereof
(substitutions, additions of chemical groups, for example, alkyl,
alkylene, hydroxylations, oxidations, and other modifications
routinely made by those skilled in the art), albumin and other
hydrophilic proteins, zein and other prolamines and hydrophobic
proteins, copolymers and mixtures thereof. In general, these
materials degrade either by enzymatic hydrolysis or exposure to
water in vivo, by surface or bulk erosion.
[0024] Bioadhesive polymers of particular interest include
bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and
J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of
which are incorporated herein, polyhyaluronic acids, casein,
gelatin, glutin, polyanhydrides, polyacrylic acid, alginate,
chitosan, poly(methyl methacrylates), poly(ethyl methacrylates),
poly(butylmethacrylate), poly(isobutyl methacrylate),
poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl
methacrylate), poly(phenyl methacrylate), poly(methyl acrylate),
poly(isopropyl acrylate), poly(isobutyl acrylate), and
poly(octadecyl acrylate).
[0025] Selection of Matrix Form and Size
[0026] In the preferred embodiment, the polymeric matrix is a
microparticle between nanometers and one millimeter in diameter,
more preferably between 0.5 and 100 microns for administration via
injection or inhalation (aerosol).
[0027] The microparticles can be microspheres, where gene is
dispersed within a solid polymeric matrix, or microcapsules, where
the core is of a different material than the polymeric shell, and
the gene is dispersed or suspended in the core, which may be liquid
or solid in nature. Unless specifically defined herein,
microparticles, microspheres, and microcapsules are used
interchangeably.
[0028] Alternatively, the polymer may be cast as a thin slab or
film, ranging from nanometers to four centimeters, a powder
produced by grinding or other standard techniques, or even a gel
such as a hydrogel. The polymer can also be in the form of a
coating or part of a stent or catheter, vascular graft, or other
prosthetic device.
[0029] Methods for Making the Matrix
[0030] The matrices can be formed by solvent evaporation, spray
drying, solvent extraction and other methods known to those skilled
in the art.
[0031] Microsphere Preparation
[0032] Bioerodible microspheres can be prepared using any of the
methods developed for making microspheres for drug delivery, for
example, as described by Mathiowitz and Langer, J. Controlled
Release 5, 13-22 (1987); Mathiowitz, et al., Reactive Polymers 6,
275-283 (1987); and Mathiowitz, et al., J. Appl. Polymer Sci. 35,
755-774 (1988), the teachings of which are incorporated herein. The
selection of the method depends on the polymer selection, the size,
external morphology, and crystallinity that is desired, as
described, for example, by Mathiowitz, et al., Scanning Microscopy
4, 329-340 (1990); Mathiowitz, et al., J. Appl. Polymer Sci. 45,
125-134 (1992); and Benita, et al., J. Pharm. Sci. 73, 1721-1724
(1984), the teachings of which are incorporated herein.
[0033] In solvent evaporation, described for example, in
Mathiowitz, et al., (1990), Benita, and U.S. Pat. No. 4,272,398 to
Jaffe, the polymer is dissolved in a volatile organic solvent. The
DNA, either in soluble form or dispersed as fine particles, is
added to the polymer solution, and the mixture is suspended in an
aqueous phase that contains a surface active agent such as
poly(vinyl alcohol). The resulting emulsion is stirred until most
of the organic solvent evaporates, leaving solid microspheres.
[0034] In general, the polymer can be dissolved in methylene
chloride. Several different polymer concentrations can be used, for
example, between 0.05 and 0.20 g/ml. After loading the solution
with DNA, the solution is suspended in 200 ml of vigorously
stirring distilled water containing 1% (w/v) poly(vinyl alcohol)
(Sigma Chemical Co., St. Louis, Mo.). After four hours of stirring,
the organic solvent will have evaporated from the polymer, and the
resulting microspheres will be washed with water and dried
overnight in a lyophilizer.
[0035] Microspheres with different sizes (1-1000 microns) and
morphologies can be obtained by this method which is useful for
relatively stable polymers such as polyesters and polystyrene.
However, labile polymers such as polyanhydrides may degrade due to
exposure to water. For these polymers, hot melt encapsulation and
solvent removal may be preferred.
[0036] In hot melt encapsulation, the polymer is first melted and
then mixed with the solid particles of DNA, preferably sieved to
less than 50 .mu.m. The mixture is suspended in a non-miscible
solvent such as silicon oil and, with continuous stirring, heated
to 5.degree. C. above the melting point of the polymer. Once the
emulsion is stabilized, it is cooled until the polymer particles
solidify. The resulting microspheres are washed by decantation with
petroleum ether to give a free-flowing powder. Microspheres with
diameters between one and 1000 microns can be obtained with this
method. The external surface of spheres prepared with this
technique are usually smooth and dense. This procedure is useful
with water labile polymers, but is limited to use with polymers
with molecular weights between 1000 and 50000.
[0037] Solvent removal was primarily designed for use with
polyanhydrides. In this method, the drug is dispersed or dissolved
in a solution of a selected polymer in a volatile organic solvent
like methylene chloride. The mixture is then suspended in oil, such
as silicon oil, by stirring, to form an emulsion. Within 24 hours,
the solvent diffuses into the oil phase and the emulsion droplets
harden into solid polymer microspheres. Unlike solvent evaporation,
this method can be used to make microspheres from polymers with
high melting points and a wide range of molecular weights.
Microspheres having a diameter between one and 300 microns can be
obtained with this procedure. The external morphology of the
spheres is highly dependent on the type of polymer used.
[0038] In spray drying, the polymer is dissolved in methylene
chloride (0.04 g/ml). A known amount of active drug is suspended
(if insoluble) or co-dissolved (if soluble) in the polymer
solution. The solution or the dispersion is then spray-dried.
Typical process parameters for a mini-spray drier are as follows:
polymer concentration=0.04 g/ml, inlet temperature=24.degree. C.,
outlet temperature=13 to 15.degree. C., aspirator setting=15, pump
setting=10 ml/min, spray flow=600 NLh.sup.-1, and nozzle
diameter=0.5 mm. Microspheres ranging in diameter between one and
ten microns can be obtained with a morphology which depends on the
selection of polymer.
[0039] Double walled microspheres can be prepared according to U.S.
Pat. No. 4,861,627 to Mathiowitz.
[0040] Hydrogel microspheres made of gel-type polymers such as
alginate or polyphosphazines or other dicarboxylic polymers can be
prepared by dissolving the polymer in an aqueous solution,
suspending the material to be incorporated into the mixture, and
extruding the polymer mixture through a microdroplet forming
device, equipped with a nitrogen gas jet. The resulting
microspheres fall into a slowly stirring, ionic hardening bath, as
described, for example, by Salib, et al., Pharmazeutische Industrie
40-11A, 1230 (1978), the teachings of which are incorporated
herein. The advantage of this system is the ability to further
modify the surface of the microspheres by coating them with
polycationic polymers such as polylysine, after fabrication, for
example, as described by Lim, et al., J. Pharm. Sci. 70, 351-354
(1981). For example, in the case of alginate, a hydrogel can be
formed by ionically crosslinking the alginate with calcium ions,
then crosslinking the outer surface of the microparticle with a
polycation such as polylysine, after fabrication. The microsphere
particle size will be controlled using various size extruders,
polymer flow rates and gas flow rates.
[0041] Chitosan microspheres can be prepared by dissolving the
polymer in acidic solution and crosslinking with tripolyphosphate.
For example, carboxymethylcellulose (CMC) microsphere are prepared
by dissolving the polymer in an acid solution and precipitating the
microspheres with lead ions. Alginate/polyethylene imide (PEI) can
be prepared to reduce the amount of carboxyl groups on the alginate
microcapsules. Table 1 summarizes various hydrogels,
concentrations, ionic baths, and stirring rates used to manufacture
them.
1TABLE 1 Preparation of Hydrogel Matrices Hydrogel dissolving bath
ionic bath stirring Hydrogel concen. pH Temp .degree. C. concen.
(w/v) rate chitosan 1.0% 5.0 23.degree. C. 3% tripoly- 170 rpm
phosphate alginate 2.0% 7.4 50.degree. C. 1.3% calcium 160 rpm
chloride alginate/ 2.0%/ 7.4 50.degree. C. 1.3% calcium 160 rpm PEI
6.0% 7.4 50.degree. C. chloride Carboxy 2.0% 7.4 50.degree. C.
10.0% lead 100 rpm methyl nitrate cellulose
[0042] Other Device Forms
[0043] Other delivery systems including films, coatings, pellets,
slabs, and devices can be fabricated using solvent or melt casting,
and extrusion, as well as standard methods for making composites.
The polymer can be produced by first mixing monomers and DNA as
described by Sawhney, et al., and polymerizing the monomers with UV
light. The polymerization can be carried out in vitro as well as in
vivo. Thus, any biocompatible glue could be also used to
incorporate the DNA.
[0044] Loading of Gene
[0045] The range of loading of the gene to be delivered is
typically between about 0.01% and 90%, depending on the form and
size of the gene to be delivered and the target tissue.
[0046] Selection of Genes to be Incorporated
[0047] Any genes that would be useful in replacing or supplementing
a desired function, or achieving a desired effect such as the
inhibition of tumor growth, could be introduced using the matrices
described herein. As used herein, a "gene" is an isolated nucleic
acid molecule of greater than thirty nucleotides, preferably one
hundred nucleotides or more, in length.
[0048] Examples of genes which replace or supplement function
include the genes encoding missing enzymes such as ad nosine
deaminase (ADA) which has been used in clinical trials to treat ADA
deficiency and cofactors such as insulin and coagulation factor
VIII.
[0049] Genes which effect regulation can also be administered,
alone or in combination with a gene supplementing or replacing a
specific function. For example, a gene encoding a protein which
suppresses expression of a particular protein-encoding gene, or
vice versa, which induces expresses of a protein-encoding gene, can
be administered in the matrix.
[0050] Examples of genes which are useful in stimulation of the
immune response include viral antigens and tumor antigens, as well
as cytokines (tumor necrosis factor) and inducers of cytokines
(endotoxin), and various pharmacological agents.
[0051] The chronic immune response to the polymeric matrix is
mediated by the action of a variety of growth factors including
epidermal growth factor (EGF), platelet-derived growth factor
(PDGF), fibroblast growth factors (FGFs), transforming growth
factors (TGF-.alpha. and TGF-.beta., interleukin-1 (IL-1), and
tumor necrosis factor (TNF). Inhibitors of these inflammatory
mediators in combination with a gene to be delivered other than the
immune inhibitor would be effective in decreasing the normal
inflammatory response directed toward the polymeric matrix. By
inhibiting the amount of encapsulation of the matrix, the effective
release would be further extended. Examples of materials which
could inhibit encapsulation include antisense mRNA to suppress
fibrin or collagen formation, inhibitors of EGF, PDGF, FGFs,
TGF-.alpha., TGF-.beta., IL-1 and TNF and anti-inflammatory agents
such as corticosteroids and cyclosporin.
[0052] Genes can be obtained using literature references or from
commercial suppliers. They can be synthesized using solid phase
synthesis if relatively small, or obtained in expression vectors,
for example, as deposited with the American Type Culture
Collection, Rockville, Md.
[0053] Selection of Vectors to be Introduced in Combination with
the Gene.
[0054] As used herein, vectors are agents that transport the gene
into the cell without degradation and include a promoter yielding
expression of the gene in the cells into which it is delivered.
Promoters can be general promoters, yielding expression in a
variety of mammalian cells, or cell specific, or even nuclear
versus cytoplasmic specific. These are known to those skilled in
the art and can be constructed using standard molecular biology
protocols. Although as demonstrated by the examples, the genes will
diffuse out of the polymeric matrix into the surrounding cells
where they are expressed, in a preferred embodiment, the genes are
delivered in combination with a vector to further enhance uptake
and expression. Vectors are divided into two classes:
[0055] a) Biological agents derived from viral, bacterial or other
sources.
[0056] b) Chemical/physical methods that increase the potential for
gene uptake, directly introduce the gene into the nucleus or target
the gene to a cell receptor.
[0057] Biological Vectors
[0058] Viral vectors have higher transaction (ability to introduce
genes) abilities than do most chemical or physical methods to
introduce genes into cells.
[0059] Retroviral vectors are the vectors most commonly used in
clinical trials, since they carry a larger genetic payload than
other viral vectors. However, they are not useful in
non-proliferating cells.
[0060] Adenovirus vectors are relatively stable and easy to work
with, have high titers, and can be delivered in aerosol
formulation. However, many people may have pre-existing antibodies
negating effectiveness and they are difficult to produce in
quantity.
[0061] Pox viral vectors are large and have several sites for
inserting genes, they are thermostable and can be stored at room
temperature. However, they cannot be transmitted from host to host
and there are some safety issues since they can enter other
cells.
[0062] Plasmids are not integrated into the genome and their life
span is from few weeks to several months, so they are typically
very safe. However, they have lower expression levels than
retroviruses and since cells have the ability to identify and
eventually shut down foreign gene expression, the continuous
release of DNA from the polymer to the target cells substantially
increases the duration of functional expression while maintaining
the benefit of the safety associated with non-viral
transfections.
[0063] Chemical/Physical Vectors
[0064] Other methods to directly introduce genes into cells or
exploit receptors on the surface of cells include the use of
liposomes and lipids, ligands for specific cell surface receptors,
cell receptors, and calcium phosphate and other chemical mediators,
microinjections directly to single cells, electroporation and
homologous recombination. The chemical/physical methods have a
number of problems, however, and will typically not be used with
the polymeric matrices described herein. For example, chemicals
mediators are impractical for in vivo use: when calcium phosphate
is used there appears to be very low transduction rate, when sodium
butyrate is used the inserted gene is highly unstable and when
glycerol is used inserted gene is rapidly lost.
[0065] Pharmaceutical Compositions
[0066] The microparticles can be suspended in any appropriate
pharmaceutical carrier, such as saline, for administration to a
patient. In the most preferred embodiment, the microparticles will
be stored in dry or lyophilized form until immediately before
administration. They will then be suspended in sufficient solution
for administration.
[0067] In some cases, it may be desirable to administer the
microparticles in combination with an adjuvant to enhance the
inflammatory response against the polymer and thereby increase the
likelihood of phagocytosis by macrophages and other hematopoietic
cells, with subsequent expression of the gene specifically within
these cells, or, in the case where the microparticles contain an
anti-cancer agent, to enhance the inflammatory reaction against the
tumor cells in combination with the effect of the anti-cancer
agent.
[0068] The polymeric microparticles can be administered by
injection, infusion, implantation, orally (not preferred), or
administration to a mucosal surface, for example, the
nasal-pharyngeal region and/or lungs using an aerosol, or in a
cream, ointment, spray, or other topical carrier, for example, to
rectal or vaginal areas. The other devices are preferably
administered by implantation in the area where release is
desired.
[0069] The materials can also be incorporated into an appropriate
vehicle for transdermal delivery as well as stents. Appropriate
vehicles include ointments, lotions, patches, and other standard
delivery means.
[0070] Targeting of Cell Populations Through Polymer Material
Characteristics.
[0071] Studies with plasmid release using PLA/PCL biodegradable
polymers indicate that the majority of transfected cells, assessed
with the .beta.-galactosidase reporter gene, are inflammatory cells
involved in the "foreign body" response. In general, non-degrading
polymers evoke a stronger inflammatory response when compared to
non-biodegrading polymers. A strong foreign body response results
in a thick layer of macrophages, fibroblasts, and lymphocytes
around the implant. Because the polymer release device relies on
diffusion for movement of its particles, a strong inflammatory
response will limit the effective distance of diffusion.
Accordingly, biodegrading polymers can be used to target
inflammatory cells due to the inability of the plasmid DNA (pDNA)
to migrate across the reactive tissue layer to the site specific
tissue. A more biocompatible material which induces a weaker
response from the host will result in a thinner layer of
inflammatory cells, enabling the released pDNA to migrate across
the inflammatory cells to the indigenous cells to be
transfected.
[0072] Incorporation of Antiinflammatories and Immune Enhancers;
Treatment of Cancers
[0073] In recent years, considerable attention has been focused on
the use of gene therapy to treat various diseases including cancer.
Generally, gene therapy for cancer therapeutics either targets the
cells of the immune system to enhance their ability to kill
malignant cells or directly targets the cancer cells to regulate
their proliferation or enhance some cellular function which will
result in a stronger activation of the immune response.
[0074] Most types of cancer are characterized by frequent relapses
during the course of treatment and the continued non-specific
and/or specific activation of the immune system resulting from gene
therapy is crucial. Second, cell targeting is a major limitation of
current vectors and implantation of a controlled release device
directly inside a tumor where the DNA is released locally is one
alternative to ex vivo therapy or the development of effective
ligand specific vectors. As indicated by the prevalence of ex vivo
therapy, targeting hematopoietic cells is especially difficult. The
histological results from the implant site in the studies described
in the examples below, reveal a substantial inflammatory response
surrounding the intramuscular implant. The well known "foreign
body" host response can be used to an advantage as this migration
of lymphocytes and antigen presenting cells raises the possibility
of directing the transfection to these specific cell
populations.
[0075] Tumors elicit both the humoral and cell-mediated immune
response, and lymphocytes, particularly cytotoxic T cells and NK
cells, as well as macrophages, are known to play a crucial role in
tumor elimination. Gene therapy for cancer treatment either targets
these cells or the malignant cells themselves. An implant releasing
naked DNA for long term functional gene transfer which can target
inflammatory cells and/or tumor cells could significantly improve
cancer therapy.
[0076] The approaches used include upregulation of class I MHC
expression, transduction of antigen presenting cells with
tumor-specific antigens, cytokine immunotherapy, transfection of
tumor cells with tumor suppressor genes and anti-sense therapy.
[0077] The malignant transformation of cells is often characterized
by a reduction of class I MHC expression leading to a severe
depression of the CTL-mediated immune response. An increase in
class I MHC expression on tumor cells could facilitate the
activation of the immune system against these altered self-cells.
Transfection of genes for cytokines such as tumor necrosis factor
(TNF) into tumor cells or tumor suppressor genes such as p53 can be
used to limit th ability of tumor cells to multiply. Anti-sense
therapy targets cell proliferation or the production of necessary
proteins such as tumor angiogenesis factor (TAF) by complementary
RNA hybridization to block transcription of specific genes.
[0078] The immune system can be activated and induced to attack
specific cells using cytokines such as Proleukin or monoclonal
antibodies. For example, cancer cells proliferate in part due to a
decreased immune response against the transformed cells. The
matrices described herein provide a means to allow recognition and
provocation of a response to cancer cells. For example, genes
coding for antigens, such as the aberrant epithelial mucin of
breast cancer, and monoclonal antibodies directed against tumor
antigens have been shown to have potential in stimulating immune
destruction of malignant cells. These genes, alone or in
combination with monoclonal antibodies, can be delivered to the
tumor sites in the polymeric matrices to achieve inhibition of the
tumor cells.
[0079] Cancer cells can also be treated by introducing chemotherapy
drug resistant genes into healthy cells to protect them against the
toxicity of drug therapy, or by the insertion of appropriate
vectors containing cytotoxic genes or blocking genes into a tumor
mass to eliminate cancer cells. In a preferred embodiment, the
immune system is specifically stimulated against antigens or
proteins on the surface of the cancer cells.
[0080] These approaches can be used in vitro and in vivo. In vitro,
the cells can be removed from a patient, the gene inserted into th
cell and the cells reintroduced into th patient. In vivo, the gene
can be directly introduced into the body either systematically or
in localized sites.
[0081] Another approach is to use suicide genes that cause cell
death when they are activated or when their product is combined
with a pharmaceutical. The primary limitation of the method is the
fact that the gene should be targeted to the cancer cell and not to
normal cell. Current approach to overcome the problem is direct
injection of the vectors into a localized area where normal cells
do not proliferate. This would be greatly facilitated using the
polymeric devices described herein. The advantages of polymeric
devices in this setting include continuous and protracted release
of the incorporated pharmaceutical. This increases the liklihood
that the intended purposes, for example, treatment of cancerous
cells, will be achieved.
EXAMPLES
[0082] The method and materials of the present invention will be
further understood by reference to the following non-limiting
examples.
Example 1
[0083] Expression of Linear and Supercoiled Plasmid DNA
Encapsulated in Polymeric Implants in Muscle Tissue of Rats.
[0084] The study described in this example confirms the feasibility
of in vivo transfections using biodegradable polyester blends to
release linear or supercoiled plasmid DNA. Although only short term
expression was studied in this study, polymer devices releasing
drugs offer the potential for sustained long term delivery of naked
DNA.
[0085] Marker genes are used to study the movement of engineered
cells containing exogenous genes, as well as the vectors and genes
introduced with the vectors, to insure that the genes remain where
they are introduced. Almost all of the initial research into gene
therapy is with marker genes. Preferred marker genes are those
whose product is innocuous and which can be readily detected by
simple laboratory tools. An appropriate marker gene is
.beta.-galactosidase (.beta.-gal), since expression is readily
detected by addition of X-gal, a substrate which yields a blue
color when the active enzyme is present.
[0086] Encapsulation of Linear and Supercoiled .beta.-gal Coding
DNA in a PLA Blend
[0087] 1 g PLA (300K) and 2 g PLA (2K) was dissolved in 10 ml of
methylene chloride and 5 drops of Span 85. The mixture was divided
into two aliquots of 5 ml and 100 ul of either circular or linear
DNA (between 1 and 2 mg/ml diluted 1:5 in buffer) was introduced
into the aliquots. Each mixture was mixed well and aliquoted into
glass vials (1 ml/vial). Between 20 .mu.g and 40 .mu.g of
.beta.-gal plasmid DNA was encapsulated in each glass vial. The
glass vials were left in the refrigerator for four days to
evaporate the methylene chloride and then lyophilized.
[0088] Implantation of DNA/PLA Pellets
[0089] Each sample was first sterilized with ethanol for 5 min and
then washed with PBS-penicillin/streptomycin for 5 min. Surgery was
done on Sprague Dawley rats. Linear DNA was implanted into the left
leg and supercoiled DNA implanted into the right. Implants were
inserted into incised muscle--either in the vastus or the
hamstring. The muscle was sutured back together and then the skin
was sutured closed. Rats were sacrificed for analysis at two
weeks.
[0090] Results
[0091] Rats were perfused with Phosphate Buffered Saline (PBS) with
2500 units of heparin followed by 3% paraformaldehyde and 0.2%
glutaraldehyde in PBS. The tissue was post-fixed with 3%
paraformaldehyd followed by 15% sucrose/PBS. Excised muscles were
cut with a cryostat and stained with X-gal.
[0092] Histology of the implant sites revealed a substantial
inflammatory response around the film at two weeks and two months.
The bulk of the .beta.-gal positive staining was localized to this
area with few muscle cells exhibiting positive staining. The cells
present around the implant probably consists of phagocytic cells,
lymphocytes and fibroblasts. As expected, transfection was more
efficient with supercoiled DNA.
Example 2
[0093] In vitro Transfection with pRSV .beta.-gal.
[0094] NIH3T3 fibroblasts were plated onto a 6 well tissue culture
dish with 1 ml of D-MEM (10% Fetal calf serum with
penicillin/streptomycin). 24 hours after plating, the cells were
transfected with pRSV .beta.-gal control plasmids as per Promega
Profection Mammalian Transfection system.
[0095] Plate 1: 10 .mu.l pRSV-Z (3.4 .mu.g) Calcium Phosphate
Precipitated
[0096] Plate 2: 30 .mu.l PRSV-Z (10.2 .mu.g) Calcium Phosphate
Precipitated
[0097] Plate 3: 10 .mu.l PRSV-Z (3.4 .mu.g) Naked DNA
[0098] Plate 4: 30 .mu.l PRSV-Z (10.2 .mu.g) Naked DNA
[0099] Plate 5: DNA/PLA
[0100] Plate 6: Control
[0101] Plate 5 with the PLA pellet was placed into the well with 4
ml of media to counter the effect of the decrease in pH. After 24
hours, the DNA/PLA pellet was removed and the media left unchanged.
At 48 hours, the cells were fixed and stained with X-Gal (1
ml/plate) overnight.
[0102] Results
[0103] The efficiency of transfection was very low. All plates
except the control well had a handfull of blue staining cells.
There was no observable differences in the number of blue cells
among the 5 plates. It was interesting to note that the plate with
the DNA/PLA had similar levels of staining as the other plates even
after the fact that half the cells had died and detached due to the
PLA degradation.
Example 3
[0104] Duration of Expression with pSV .beta.-gal DNA Encapsulated
Into PLA Blends.
[0105] In vitro Release of Plasmid DNA
[0106] pSV .beta.-gal was amplified in HB101 and purified with
Qiagen's Mega Prep. 500 .mu.l of plasmid in Tris-EDTA buffer (67.5
.mu.g) was lyophilized and resuspended into 100 .mu.l of sterile
dH.sub.2O and incorporated into PLA. 0.05 g PLA (2K) and 0.05 g
(300K) was dissolved in 1 ml of methylene chloride and 1 drop of
Span.TM. 85. After the polymer was in solution, 100 .mu.l of
plasmid (67.5 .mu.g) was added to the mixture and vortexed for 15
sec. The resulting film was left in a refrigerator overnight and
subsequently lyophilized overnight.
[0107] This film was incubated with 1.0 ml of TE buffer at
37.degree. C. under gentle agitation and sample supernatants tested
at 24 hours and at 4 days for the presence of released DNA. DNA was
assayed by agarose gel electrophoresis on the supernatants.
[0108] The results based on the gel of the supernatant after 24
hours of incubation show that a substantial amount of plasmid was
released. After 4 days, the results indicate that there was a first
phase of release due to the diffusion of plasmid molecules which
are close to the surface of the device followed by a slower release
at 4 days due to the low degradation rate of the polymer which was
too low to be measured.
[0109] In vivo Transfection Levels
[0110] 3 mg PLA (2K) and 1 mg PLA (100K) were dissolved in
methylene chloride (0.25 ml). 1 drop of Span.TM. 85 and 20 .mu.l of
plasmid (20 .mu.g) was added to the solution and homogenized for 1
minute. This solution was air dried in a glass vial for 3 hours in
a sterile hood. The brittle film was ground into fine granules and
pressed into a pellet form. Three of these DNA containing pellets
were made as well as three control pellets without DNA. All pellets
were lyophilized overnight to extract residual solvents.
[0111] Three rats received DNA/PLA in their left hamstring and
control/PLA in their right hamstring. Pellets were inserted into
incised hamstrings and the muscles closed with 6-0 Vicryl. Three
rats received an injection of pSV .beta.-gal plasmids (20 .mu.g in
100 .mu.l of TE buffer) over a minute long period in their left leg
and 100 .mu.l of plain TE buffer in their right leg as controls.
The site of injection was marked with suture.
2 Rat ID Left Right Implant Duration R112 DNA/PLA Control/DNA 1
week R110 DNA/PLA Control/DNA 5 weeks R111 DNA/PLA Control/DNA 10
weeks R115 DNA/buffer Control/buffer 1 week R114 DNA/buffer
Control/buffer 5 weeks
[0112] Rats were perfused with PBS/heparin, followed by 4%
paraformaldehyde, and post-fixed in 4% paraformaldehyde followed by
15% and 25% sucrose/PBS. Excised muscles were cut with a cryostat
and stained with X-Gal.
[0113] Results
[0114] In vitro release studies indicate that plasmid DNA can be
incorporated into polymers without degradation through
manufacturing processes and released in functional form for
possible uptake by surrounding cells.
[0115] In vivo studies reveal that with a 20 .mu.g loading of DNA
into the polymer, there is substantial transfection of inflammatory
cells at 1 and 5 weeks as confirmed by X-gal staining and
immunoblotting. At 10 weeks, there was no difference in staining
intensity between the control PLA and DNA/PLA. This is believed to
be due to the result of the low loading (20 .mu.g) of the polymer
such that after one week the release rate was below half maximal
levels. Investigators using direct injection use doses in the 100
.mu.g range to see their effects. A higher initial loading, which
will lead to continued release of higher amounts of DNA from
polymers, should prolong transfection durations. Rats injected with
20 .mu.g of DNA in solution showed no transfection at 1 and 5
weeks.
Example 4
[0116] Comparison of Plasmid DNA Release From Biodegradable and
Non-degrading Polymers.
[0117] Release of plasmids from biodegradable and non-degradable
polymer was compared to test the feasibility of targeting either
inflammatory cells or tissue specific cells by selection of polymer
material. Plasmid DNA was incorporated into a non-degradable
elastomer, ethylene vinyl acetate copolymer (EVAc) and implanted
into the same site in different animals as PLA/PCL implants. EVAc
is a very biocompatible polymer which can be manufactured into a
microporous structure through which DNA can diffuse into the
surrounding tissue.
[0118] Encapsulation of pRSV .beta.-gal into Polymers.
[0119] pRSV .beta.-gal in HB101 was purchased from the ATCC
(American Type Culture Collection, Rockville, Md.). The plasmids
were grown and purified with Promega's Maxi Prep. 1 ml of a 0.1%
solution of ELVAX40 (Dupont) in methylene chloride was vortexed
with 645.2 .mu.l of pRSV .beta.-gal (200 .mu.g), frozen in liquid
nitrogen and lyophilized. The resulting mixture was extruded at
55.degree. C. into a rod shaped form.
[0120] PLA (2K) and PCL (112K) were dissolved in methylene chloride
in a 3:1 ratio and 80 mg of the polymer vortexed with 322.6 .mu.l
of pRSV .beta.-gal (100 .mu.g). The mixture was left in the
refrigerator for 2 days and lyophilized.
[0121] Implantation of the Polymers.
[0122] The EVAc/DNA and PLA/DNA were implanted into rat hamstrings
along with their control on opposite sides and sacrificed at 2
weeks.
[0123] Results.
[0124] Histological staining with X-gal reveals positive staining
of muscle cells as well as inflammatory cells in close proximity to
the EVAc polymeric implant at two weeks post-implantation. In
comparison, the PLA/PCL implant reveals positive staining of mostly
inflammatory cells only, in accordance with the earlier data
regarding biodegradable polymers.
[0125] Thus the selection of a biodegradable or non-degradable
polymer implant can be used to target delivery to inflammatory
cells or tissue cells (for example, muscle). Comparison of PLA/PCL
and the EVAC implants illustrates the different transfected cell
populations. Specifically, the PLA/PCL implant results in almost
exclusive transfection of inflammatory cells while the EVAc implant
results in a large number of transfected muscle cells.
[0126] Modifications and variations of the method and compositions
of the present invention will be obvious to those skilled in the
art from the foregoing detailed description. Such modifications and
variations are intended to come within the scope of the following
claims.
* * * * *