Note: Descriptions are shown in the official language in which they were submitted.
W094/~738 21~ ~ 8 7 8 PCT~S94/~9
TITLE
ENCAPSULATION OF NUCLEIC ACIDS
~ITH CONJUGATES THAT FACILITATE AND
TARGET CELLULAR UPTAKE AND GENE EXPRESSION
5~IF.T.~ OF THF I~VFNTION
The present invention relates to the controlled
release of nucleic acids (DNA, RNA, synthetic
oligonucleotides or derivatives thereof) conjugated or
in combination with proteins, antibodies, or other
molecules within slow release biodegradable polymeric
microparticles. The present invention also relates to
the use of encapsulated genes for qenetic
transformation.
R~CKGROUND OF THF INVENTION
15One of the rate-limiting steps in biotechnology is
the insertion of genetic material into cells and
tissues. To date, known methods have not provided
satisfactory means to facilitate the controlled uptake
and integration of genetic material into living cells of
animals and plants for the useful expression of
exogenous genes.
Various efforts have been directed at improving the
uptake of exogenous nucleic acids into specific cells.
Several molecules and receptors have been shown to
faci~itate nucleic acid uptake into specific cells. For
example, asialoglycoprotein increases uptake into liver
cells (Wu et al., J. Biol. Chem., 2b2:4429-4432 (1987)?,
transferrin-polylysine adenovirus facilitate uptake into
epithelial cells (Curiel et al., Proc. Natl. Acad. Sci.
USA, 88:8850-8854 (1991)), and viral particles such as
herpes virus are transported into neural cells (Geller
et al., Science 241:1667-1669 (1988)). Anti~odies that
bind to specific cellular ligands can also facilitate
uptake to specific cells and tissues. In addition,
liposomes with receptor ligands have been used to
W094l~738 ~ ,t 2 ~ 6 ~ 8 7 8 PCT~S94/04~9
transfer DNA to cells ~Malone et al., Proc. Natl. Acad.
Sci. USA, 86:6077-6081 (1989)). These preferential and
specific uptake molecules bind to specific cell-surface
receptors and are internalized by the receptor mediated-
endocytosis pathway.
Various chemical modifications have been performed
on nucleic acids which help promote their up-take and
subsequent expression by cells. It has been shown
(Huckett et al., Biochem. Pharmacol., 40:253-263 (1990))
that when DNA was non-covalently bound to a chemically
modified albumin cross-linked to insulin, the
trimolecular complex was bound by the insulin receptor
on HepG2 cells, taken into the cell by receptor-mediated
endocytosis, transported to the nucleus and expressed in
the form of mRNA transcription and protein translation.
Recently, molecules that target integration into
specific sites on the chromosome have been identified.
Moreover, DNA-binding proteins and other molecules can
promote transcription.
What these methods lack is the means to protect the
components from degradation. A solution to this problem
would not only protect the components but also permit
slow-release of the encapsulated nucleic acids and
ligands.
Gene Delivery Methods
Existing technologies for transporting genetic
material into living cells involve methods for chemical
uptake, retroviral infection, and physical insertion
including particle bombardment. Several of these
methods can be improved with use of encapsulated nucleic
acids with conjugates as proposed herein. Particle-
mediated bombardment of DNA to plant, animal and
microbial cells as well as to living animals have been
reported IFitzpatrick-McElligott, Bio/Technology
10:1036-1040 (1992), Klein et al, Bio/Technology
21~087~
WOg4~3738 ^ PCT~S94/04~9
10:286-291. (1992), Williams et al. Proc. Natl. Acad.
Sci. USA. 88:2726-2730 (1991)].
Currently known methods have the disadvantage of
permanent germ line integration of genetic material
which in certain cases is not desirable. Once a gene
has integrated into the cell germ line, as is the case
with retrovirus-mediated gene transfer, it is usually
there until the cell dies. Patients not requiring
permanent gene replacement cannot be treated by
retroviral gene therapy. Another issue is safety.
Retroviruses are oncogenic viruses and are inherently
dangerous for use in the general human population.
Currently, protein injections are used for the
treatment of some diseases. Protein injections can
result in an uneven availability, leading to potentially
toxic doses immediately after injection, with
insufficient amounts later on. A method of somatic
transformation with slow-release genetic material to
allow sustained production and delivery of proteins and
peptides is needed.
Biodegradable Microparticles
The use of biodegradable microparticles containing
drugs as a slow release delivery system is known. U.S.
Patents Nos. 4,389,330 and 4,542,025 disclose a variety
of microparticles, or microcapsules, their preparations
and their usage.
European Patent Application 248,531 discloses RNA
and/or DNA or antisense RNA in microcapsules for the
induction of interferon production, which is said to be
a potential inhibitor of viral replication. However,
the European patent application fails to provide a
method for uplake and delivery to the intracellular
cytoplasm of the cells. Therefore, use of the method as
outlined by the European patent application would not be
wo94l23738 2160878 rcTluss4lo4~3s
sufficient for the use of genetic material and gene
expression.
Encapsulation of genetic material would protect the
nucleotides from enzymatic degradation before they are
released. Controlled release of genes would also reduce
lethality to the organisms by allowing controlled
expression of product.
SUM~RY OF T~F. I~V~TION
The present invention is a microparticle
composition suitable for the controlled release of a
nucleic acid to a target cell, the microparticle
comprising a microparticle composition suitable for the
controlled release of a nucleic acid to a target cell,
the microparticle comprising:
~a) a nucleic acid comprising synthetic or
natural DNA or RNA exogenous or native to a target cell,
the nucleic acid conjugated by way of chemical bonds
with promoting material which promotes the upta~e or the
transport to the nucleus, or expression of the nucleic
acid in the cell, the molecules selected from the group
consisting of glycoproteins, lipoproteins,
nucleoproteins and peptides, hormones, antibodies,
growth factors, nucleic acid binding factors,
proteinaceous cellular ligands, glycolipids,
peptidoglycans, lectins,.fatty acids, phospholipids,
glycolipids, triglycerides, s~eroid hormones,
cholesterol, single stranded or double stranded RNA,
single stranded or double stranded DNA, and
intercalating agents, the nucleic acid present in an
amount of about 0.U001 wt % to 50 wt % based on the
parts of nucleic acid per weight of an encapsulating
polymeric matrix of element (b);
(b) a biocompatible, biodegradable polymeric
matrix encapsulating the nucleic acid of element (a),
the polymeric matrix selected from the group consisting
W094/~738 216 0 8 7 8 PCT~S94/04~9
of poly-d, L-lactic acid, poly-L-lactic acid,
polyglycolic acid, copolymers of mixed d,L-lactic acid
and glycolic acid, copolymers of L-lactic acid and
glycolic acid, copolyoxalates, polycaprolactone,
poly(lactic acid-caprolactone), poly(glycolic
acid-caprolactone), casein, albumin, and waxes; and
(c) the microparticle ranging in diameter from 1
to 500 microns. Additionally, the invention encompases
a microparticle composition suitable for the controlled
release of a nucleic acid to a target cell, the
microparticle comprising a microparticle composition
suitable for the controlled release of a nucleic acid to
a target cell, the microparticle comprising:
(a) a nucleic acid comprising synthetic or
natural DNA or RNA exogenous or native to a target cell,
the nucleic acid conjugated by way of chemical bonds
with promoting material which promotes the uptake or the
transport to the nucleus, or expression of the nucleic
acid in the cell, the molecules selected from the group
consisting of glycoproteins, lipoproteins,
nucleoproteins and peptides, hormones, antibodies,
growth factors, nucleic acid binding factors,
proteinaceous cellular ligands, glycolipids,
peptidoglycans, lectins, fatty acids, phospholipids,
glycolipids, triglycerides, steroid hormones,
cholesterol, single stranded or double stranded RNA,
single stranded or double stranded DNA, and
intercalating agents, the nucleic acid present in an
amount of about 0.0001 wt % to 50 wt % based on the
parts of nucleic acid per weight of an encapsulating
polymeric matrix of element (b);
tb) a biocompatible, biodegradable polymeric
matrix encapsulating the nucleic acid of element (a),
the polymeric matrix selected from the group consisting
of poly-d, ~-lactic acid, poly-L-lactic acid,
2 ~ 6 0 8 7 8
W094/~738 ~ PCT~S94tO4~9
polyglycolic acid, copolymers of mixed d,L-lactic acid
and glycolic acid, copolymers of ~-lactic acid and
glycolic acid, copolyoxalates, polycaprolactone,
poly(lactic acid-caprolactone), poly(glycolic
acid-caprolactone), casein, albumin, and waxes; and
(c) the microparticle ranging in diameter
from 1 to 500 microns. In the first case, the promoting
material is directly conjugated to the nucleic acid. In
the second case, the promoting material co-exists with
the nucleic acid within the polymeric matrix. Both of
the microparticle compositions described above may
further comprise an inert particle as the core of the
microparticle, the inert particle made of tungsten,
gold, platinum, ferrite, polystyrene, or latex.
The invention includes a method for preparing
nucleic acids in a size effective for cellular or tissue
insertion to effect gene expression in plants and
animals comprising forming a controlled release,
biocompatible biodegradable microparticle comprising one
of the two microparticle compositions set out above.
The method described above may further include
coating the polymeric matrix containing the nucleic
acids with the promoting material to promote the uptake
of the nucleic acid into the cell or the transport of
the nucleic acid to the nucleus.
The invention also includes a method for the
controlled delivery of an exogenous or native gene into
the cells of plants or animals to effect gene
expression, the method comprising:
(a) encapsulating a genetic construct
exogenous or native to a target cell within a
biocompatible, biodegradable polymer matrix, the
encapsulating step forming a microparticle composition;
2 1 6 0 8 7 ~
W094l~738 PCT~S94/04~9
(b) containing the microparticle composition
of step (a) in a dosing device suitable for delivery of
the microparticle to a target cell;
(c) discharging the microparticle composition
of step (a) into a plant or animal from which site the
genetic construct contained within the microparticle
composition can interact with the nucleus of a target
cell upon degradation of the polymer matrix.
The invention uses methods of direct delivery of
microparticles either by injection, particle bombardment
or other methods to cells and tissues either in culture
or in the living animal or plant.
RRIF.~ D~SCRIPTION OF THF DRAWINGS
Figure 1 shows the microcapsules with the genetic
material stained with DAPI (4',6-diamididino-2-phenyl-
indole) and tungsten microparticle of >1 ~m in size.
Black arrows indicate the blue DAPI stained genetic
material. The dark tungsten core is indicated by white
arrows.
Figure 2 is a scanning electron micrograph showing
the size distribution and surface qualities of the
microencapsulated particles. The microparticles contain
tungsten and plasmid DNA and herring sperm DNA.
Figure 3 is an agarose gel showing the amplified
gene after release into the solution at the specifiedtimes sampled. Lane 1 show size markers, lane 2 contain
no DNA. Lanes 3-7 show the amplified gene released into
the solution after 1, 3, 4, 5.5, and 7 hours. Lane 8
shows measurable release after 72 hours. Lane 9
indicates the amplified ~-galactosidase coding region
from the plasmid DNA.
Figure 4 shows the expression of the ~-glucuroni-
dase gene in plant cells. Four days after bombardment
of cauliflower with microencapsulated genetic material
the cauliflower tissue is placed in a reaction buffer
.
W094/~738 2 1 6 ~ 8 7 8 PCT~S94/04~9
containing X-glucuronic acid (the enzyme substrate).
Arrows show the expected ~lue staining demonstrating
gene expression in plant cells with microencapsulated
genes.
Figure 5 shows stable clone of transformed animal
cells, indicated here with a white arrow. Chinese
hamster ovary cells were bombarded with a gene construct
containing ~-galactosidase and a neomycin resistance
coding sequences. These cells, after bombardment, were
selected for neomycin resistance for four weeks.
Surviving cells demonstrate neomycin resistance, i.e.,
the activity of introduced gene. These cells multiply
to form a clone, each cell carrying the transgene. All
cells of the clone also show ~-galactosidase activity
another gene construct in the introduced plasmid DNA.
Several clones were observed after four weeks in
selection media.
Figure 6 is an agarose gel showing the amplified
gene in three clones (Lanes 3-9) after four weeks
selection in neomycin media. Lane 1 shows the molecular
weight size markers. Lane 2 shows the amplified
~-galactosidase coding region from the plasmid DNA, a
positive control marker. Lanes 3-9 show amplified DNA
from transformed clones. The amplification of the DNA
was done using the polymerase chain reaction. The
primers were designed to amplify a sequence in the
~-galactosidase coding region. Lane 10 contains DNA
from C~O cells which were not bombarded with exogenous
DNA. Lane 11 is a negative control without ~-gal DNA.
DFTAILED D~SCRIPTION OF THE INVF~TION
The proposed invention is a safe and effective
method ~or the controlled delivery of genetic
information to the intracellular cytoplasm of humans and
other animals for a finite period of time. In the
method of the present invention, nucleic acids in
W094/~738 - 21 6 0 8 7 8 PCT~S94/04~9
combination with cellular ligands are delivered to the
cells of animals and plants by known means, encapsulated
in a non-toxic, biodegradable polymer matrix. The
method solves a persistent problem in genetic
engineering by permitting controlled uptake and
prolonged gene expression in the cells and tissues of
animals, microbes and plants. Additionally, the
invention protects the encapsulated nucleic acids and
ligands from enzymatic degradation. Controlled release
of genetic material will reduce lethality to the
organism by controlling expression of the gene product.
Used with known technologies for transporting genetic
material into living cells, the invention extends their
effectiveness. Very importantly, the invention provides
the significant utility of enabling the genetic
transformation of somatic cells without germ line
integration. Somatic transformation with controlled-
release genetic material will allow sustained production
and delivery of proteins and peptides to cells.
The present invention uses encapsulated genetic
material consisting of a promotor and/or regulatory
region and coding region of specific nucleotide sequence
for the purposes of obtaining gene expression products,
transgenic organisms and for gene therapy.
The following definitions of biological and genetic
terms will be useful in understanding this invention:
The term "administered" means any method of
delivering the nucleic acid-containing microparticles of
the invention to an animal, such as, for example,
parenteral (intravenous, intramuscular, or subcutaneous)
administration or by the particle delivery method.
The term "animal" as used herein in its usual
biological annotation, and encompasses all species of
animals large enough to be treated, particularly human,
W094~3738 `J ~ - 5 ~ 2 1 6 0 ~ 7 8 PCT~S94/04~9
food animals, and mammals in general; birds, pets, fish
and the like.
The term ~'biocompatible~ may be defined as non-
toxic to the human body, non-carcinogenic and non-
inflammatory in body tissues.
The term "biodegradable" means that the polymeric
material degrades by bodily processes to products
readily disposable by the body, and do not accumulate
excessively in the body. The biodegraded products also
should be biocompatible with the body in the sense that
the polymeric matrix is compatible with the body.
The term "control" and "inhibit" as used herein as
applied to an illness mean the prevention, curing,
arrest, or other beneficial pharmacological effect on
the illness.
The term "controlled-release" as used herein with
respect to microcapsules of the present invention means
that the nucleic acid active ingredient is released from
the microcapsule polymeric matrix over an extended
period of time so as to give continuing or delayed
dosage to the treated subject. The controlled-release
period can be from 1 to 500 days and preferably is from
3 to 60 days.
"DNA sequence" is a linear sequence comprised of
any combination of the four DNA monomers, i.e.,
nucleotides of adenine, quanine, cytosine and thymine,
which codes for genetic information, such as a code for
an amino acid, a promoter, a control element (enhancer,
=nuclear recognition element, etc.) or other gene
=30 product. A specific DNA sequence is one which has a
known specific function, e.g., codes for a particular
polypeptide, a particular genetic trait or affects the
expression of a particular phenotype.
"Exogenous genetic material" is genetic material
not o~tained from or does not naturally form a part of
.
~ W094l~738 ~ Z 1 6 0 8 7 8 PCT~S94tO4~9
the specific germ cells or gametes which form the
particular zygote which is being genetically
transformed.
"Gene" is the smallest, independently functional
unit of genetic material which codes for a protein
product or controls or affects transcription and
comprises at least one DNA sequence.
"Dosing devioe" is a vehicle for introducing or
administering the microparticle to an animal or plant
cell.
"Genetic material" is a material containing any DNA
sequence or sequences either purified or in a native
state such as a fragment of a chromosome or a whole
chromosome, either naturally occurring or synthetically
or partially synthetically prepared DNA sequences, DNA
sequences which constitute a gene or genes and gene
chimeras, e.g., created by ligation of different DNA
sequences.
"Phenotypic expression" is the expression of the
code of a DNA sequence or sequences which results in the
production of a product, e.g., a polypeptide or protein,
or alters the expression of the zygote's or the
organism's natural phenotype.
The term "food animal" means any animal that is
consumed as a source of protein in the diet of humans or
other animals. Typical food animals include bovine
animals, for example cattle; ovine animals, for example
sheep; porcine, for example pigs; fowl, for example
chickens and turkeys; rabbit and the like.
"Illness" as used herein means a malady to the
species caused by internally or externally originating
entities. An illness may be localized, such as some
tumors, or it may be wide-spread throughout the animal
body as in many viral diseases. Illnesses of particular
3~ significance to the present invention are cancer and
W094/~738 .~! ~ t ~ 1 ~ 0 8 7 8 PCT~S94/~9
virally caused illnesses. The term ~locale of the
illness" refers to a localized illness and means the
zone location of the illness.
The terms ~microcapsule" or "microparticle" as used
herein mean either solid or of the reservoir type
particles which contain an active agent, herein genetic
material, either in solution or in crystalline form.
The active agent is dispersed either or dissolved within
the polymer which serves as the matrix of the particle,
or is contained within the polymer in reservoir fashion
with polymer serving as the outer wall.
CO~IPOSITIO~--GF.NF.TIC MATFRIAT.
The invention is directed toward the controlled-
release of exogenous, often chimeric, genetic constructs
into animal and plant cells. Such gene constructs would
normally include a coding region for transcription of
the protein or product, together with regulatory
se~uences. Regulatory regions may ~e promoter sequences
sufficient to initiate transcription and terminator
sequences that indicate the end of the product. The
nucleic acids useful in the process and compositions of
the present invention are, in general, recombinant or
synthetic molecules. The present invention does not use
the mechanism of interf~ron induction disclosed in EPA
No. 248,531 to inhibit cancer and viral illness.
COMPOSI~ION-CO~JUGATF.S
Promoting materials promote the upta~e or transport
to the nucleus, or expression of the nucleic acid in the
cell. These molecules are selected from the group
consisting of glycoproteins, lipoproteins,
nucleoproteins and peptides, hormones, antibodies,
growth factors, nucleic acid ~inding factors,
proteinaceous cellular ligands, glycolipids,
peptidoglycans, lectins, fatty acids, phospholipids,
= 35 glycolipids, triglycerides, steroid hormones,
. ~160878
W094l~738 ~ PCT~S94/~9
13
cholesterol, single stranded or double stranded RNA,
single stranded or double stranded DNA, and
intercalating agents, the nucleic acid present in an
amount of about 0.0001 wt % to 50 wt % based on the
parts of nucleic acid per weight of an encapsulating
polymeric matrix of element (b).
In a preferred embodiment, nucleic acids containing
new genetic information are conjugated to a ligand which
binds to a cell-surface receptor with high specificity
on the cell-type in which one wishes to express the
gene. Ligands can be coupled to DNA by several methods.
Gene transfer by means of receptor-mediated endocytosis
can be accomplished by forming bifunctional molecular
conjugates consisting of a binding ligand for a cell-
surface receptor that is covalently linked to a DNAbinding moiety. Such ligands can be targeted to
specific cells. For example, to target hepatic cells, a
galactose-terminal (asialo-)glycoprotein, the
asialoorosomucoid ligand is covalently linked to poly-L-
lysine. The conjugate is then complexed in a 2:1 molarratio to the plasmid. Asialoglycoprotein is recognized
by cell-surface asialoglycoprotein receptors unique to
the hepatic cell type (see Wu et al., J. Biol. Chem.,
262:16985-16987 (1988)). When the cell-surface receptor
recognizes the ligand, the exogenous DNA that is
complexed to the DNA binding domain is co-transported
into the cells.
Another method for DNA conjugation uses polycation-
transferrin conjugates in a complex with DNA to
introduce genes into cells. In a typical complex
formation reaction 10 ~g of transferrin-polylysine or
transferrin-protamine conjugate in 250 ~L of H2O is
added to 3 ~g of plasmid DNA contained in 250 ~L of
0.3 M NaCl (while agitating). After 30 min at room
temperature the complex is formed and can be
W094t~738 ~ 1 G 0 8 7 ~ PCT~Sg4/04~9
14
microencapsulated. By coupling the. natural iron-
delivery protein transferrin to the DNA-binding
polycations, polylysine or protamine, a protein
conjugate is created that ~inds nucleic acids and
carries them by endocytosis into the cell during the
normal transferrin cycle (Wagner et al., Proc. Natl.
Acad. Sci. USA., 87:3410-3414 (199Oj). Without
microencapsulation, endocytosis into the lysosomes,
lysosomal enzymes, proteases and nucleases hydrolyze and
destroy the DNA.
The method of the present invention protects the
DNA and conjugate by encapsulating the complex in a
biodegradable polymeric matrix. In addition, co-
encapsulation of the complex with chloroquine or
adenovirus facilitates breakdown of the lysosomes and
allows release of the intact DNA into the intracellular
cytoplasmic compartment and access to the nucleus.
Non-covalent complexing of DNA to ligand is
achieved by modifying the protein with a water-soluble
carbodiimide, N-ethyl-N'; (3-dimethylaminopropyl)
carbodiimide hydrochloride (CDI) under conditions which
allow the formation of ~asic N-acylurea moieties. The
resulting N-acylurea protein interacts to form salt
bridges with the phosphodiester backbone of the DNA [see
Huckett et al., Biochemical Pharmacoloay, 40:253-263
(1990)~. Another method involves derivitization of the
nucleic acid 5' terminus to form a hydrazide
intermediate which can be coupled with aldehyde-modified
proteins (see Ghosh et al., Anal. Biochem., 178:43-51
(1989)).
Various chemical modifications to either 3'- and/or
5'-termini or the individual nucleic acid ~ases have
been performed on DNA. Some of the chemical moieties
introduced are ~luorescent groups: the acridine
derivativesi bathophenanthroline-Ru(II) complexes
~ WO 94/23738 ~1 6 0 8 7 8 PCT/US94/04239
DANSYL, MANSYL, AEDANS-dUTP. These moieties are
generally attached through thio- or amino- linkages to
terminal hydroxyl or phosphate groups, or to the
specific bases. Other molecules which have been
attached to DNA include intercalators (e.g., acridine,
phenazium); photochemically activated cross-linking (the
psoralens) or cleaving (e.g., methylporphyrin XXI)
agents; alkylating agents (e.g., chloroal3cylaminoaryl);
redox active nucleic acid cleaving groups (e.g.,
10-Cu(II)-phenanthroline).
DNA end-labelled with biotin can form complexes
with antibodies or enzymes through an avidin biotin
complex allowing recognition of cell-surface and nuclear
membrane transport proteins. Such transport molecules
facilitate up-take of the DNA.
Lipophilic moities such as cholesterol, and long-
chain alkyl groups are generally attached at the 3' or
5' termini (see Letsinger et al. (1989) PNAS86:6553) .
Liposomes containing the DNA can be prepared by
combining 30 ~lg DNA and 100 ~L of N- [1-(2,3-dioley-
oxy)propyl]-N,N,Ntrimethyl-ammonium chloride (DOTMA) and
dioleoyl phosphatidylethanolamine in serum free media
[see Nabel et al., Science, 299:1285-1288 (1990)].
These lipophilic modifications increase the
hydrophobicity of the DNA, promoting enhanced uptake
through the cell membrane.
The method of this invention will protect the
complex and its components and allow prolonged release
of the complex by microencapsulation of the DNA
conjugates in a biodegradable polymer matrix.
M~THOD-MICROE~CAPSULATION
By adjusting the composition of the matrix polymer,
the rate of release of the drug can be predetermined and
targeted for a particular application (see Lewis in
"~3iodegradable Polymers as Drug Delivery Systems", Eds.
W0941~738 ;2 1~n 8 7 8 PCT~S94104~9
16
M. Chasin and R. Langer (1990)). The genetic material
will be available and released into the animal ~or an
extended length of time as the matrix degrades, and the
matrix will protect the unexposed gene from degrading
before it has had an effect. Because the polymer used
in the method of the invention is biodegradable, all of
the entrapped genetic material can be released into the
animal. The target cells of humans or animals would
internalize the nucleic acid-ligand conjugate by
receptor-mediated endocytosis and the nucleic acid would
be transported to the nucleus and expressed. The
duration of action can be controlled by manipulation of
the polymer composition, polymer:drug ratio and
microsphere size.
The present invention offers the advantage of
durations of action ranging from only 30 to 60 days to
more than 250 days depending upon the type of
microsphere selected. The delivery system enables
introduction of new genetic information into humans or
other animals with nucleic acids in a sustained delivery
formulation to promote either permanent or transient
gene expression and/or to knock out of an existing
cellular sequence.
The compositions of the present invention are
microparticles (microcapsules), prepared by a
conventional technique, having a biocompatible,
biodegradable polymeric matrix with nucleic acid
distributed within the matrix. The microparticles can
be of any conventional type and may include other
pharmaceutically effective ingredients, such as an
antibiotic, vaccines, and/or other conventional
additives. The formulations of the present invention
contain nucleic acid dispersed in a microparticle matrix
material.
W094~738 ~ PCT~S94/04~9
17
The preferred construction of microcapsules of the
invention are described in U.S. Pat. No. 4,389,330, U.S.
Pat. No. 4,919,929, and U.S. Pat No. 4,530,840. The
microparticles of the invention are composed of a
polymer which is, preferably, an aliphatic polyester
such as either a homopolymer or copolymer of lactic or
glycolic acids. Other degradable polymers may be used,
such as, for example, polycaprolactone, polydioxonene,
polyorthoesters, polyanhydrides, and natural polymers
including albumin, casein, and waxes. The amount of
nucleic acids incorporated in the microparticles usually
ranges from less than 0.0000~ wt% to as high as 75 wt%,
preferably 0.0001 nucleic acid to 50 wt% of the polymer.
By "weight %" is meant "parts of nucleic acids per parts
of polymer by weight". For example, 10 wt% would mean
10 parts nucleic acids per 90 parts polymer by weight.
The polymeric matrix material of the microparticles
in the present invention must be biocompatible and
biodegradable polymeric material. Suitable examples of
polymeric matrix materials include poly-d, L-lactic
acid, poly-L-lactic acid, polyglycolic acid, copolymers
of mixed d,L-lactic acid and glycolic acid, copolymers
of L-lactic acid and glycolic acid, copolyoxalates,
polycaprolactone, poly(lactic acid-caprolactone),
poly(glycolic acid-caprolactone), casein, albumin, and
waxes.
The molecular weight of the polymeric matrix
material is of some importance. The molecular weight
(MW) should be high enough so that it forms satisfactory
polymer coatings, i.e., the polymer MW is proper for the
polymer to be a good film former. Usually, a
satisfactory molecular weight is greater than 5,000
daltons. The various film-forming polymer compositions
have molecular weights readily determined by known
techniques. Also, the polymer molecular weight also
I' t ~
W094/~73$ 2 1 6 0 8 7 8 PCT~S94104~9
18
plays a significant role (along with composition,
purity, optical form, etc.) in rate of degradation. In
general, the higher the MW, the slower the degradation.
Average molecular weights of about 5,000 to 500,000 are
preferred.
Nucleic acids can also be released from the
particle by leaching through the polymer matrix, with
the nucleic acids being released before the polymer is
significantly degraded or simultaneously with polymer
degradation. By an appropriate selection of polymeric
material, a microparticle formulation can be made such
that the resulting microparticles exhibit two phases of
release properties. The selection of polymers and
manipulation of nucleic acids/polymer ratio are useful
in affording multiphasic release patterns.
The microparticle products of the present invention
can be prepared by any process capable of producing
microparticles in a size range l ~m to 500 ~m acceptable
for use in an injectable composition. Generally,
microencapsulation processes are classified according to
the principal types: tl) phase-separation methods
including aqueous and organic phase separation
processes, melt dispersion and spray drying; (2)
interfacial reactions including interfacial
polymerization, in situ polymerization and chemical
vapor deposition; (3) solvent extraction method; and (4)
physical methods, including fluidized-bed spray coating,
multi- and single-orifice centrifugal coating,
electrostatic coating and physical vapor deposition.
A preferred method preparation is the method
described in U.S. Pat. No. 4,919,929. Phase separation
methods, as the term implies, rely on differential
solubility characteristics that cause a wall- or shell-
forming matrix material to separate from solution or
3~ suspension and deposit around particles or droplets of
'? .. I' ''
W094/~738 216 0 8 7 8 PCT~S94/04~9
19
the substance to be encapsulated. The separation,
itself, may be brought about physically, as by the
addition of a non-solvent or by a change in temperature,
or chemically, as by a change in pH.
Organic phase-separation processes (see U.S. Pat.
No. 4,919,929) usually employ a dispersion or an
emulsion of the nucleic acids in a solution or a high-
molecular-weight polymer in an organic solvent. To this
mixture is added a non-solvent or liquid polymer that
causes the high-molecular-weight polymer to separate
from solution and collect as a shell around the
suspended therapeutic agent(s). The shell, still
swollen with solvent, is then hardened by a further
addition of non-solvent or by some other process that
strengthens the shell and improves the barrier
properties, controlling release by its nucleic acids
permeability and/or degradation rate.
Typically in the above described organic phase-
separation process, an aqueous solution or suspension of
a lipophobic antigen is added to a non-aqueous solution
of a suitable matrix polymer, and the mixture is
agitated to cause the formation of a water-in-oil
emulsion. Depending upon its solubility in water, the
agent may be present at a concentration of 0.1 to 50% in
the aqueous phase, which may be 0.1 to 20% by weight of
the total mixture. The external organic phase may
contain 5 to 10% of the matrix polymer. Usually,
however, the ratio of agent in the internal phase
(aqueous solution or suspension) to polymer is 2:1 to
1:4.
An aqueous phase separation process (see U.S. Pat.
No. 4,919,929) employs a dispersion or an emulsion of a
water-insoluble therapeutic substance in an aqueous
solution or dispersion of a polymer. The polymer is
caused to separate as gel particles; these collect
f ~ D87 8
wOg4/~738 PCT~S94/04~9
around the therapeutic agent to form a shell; the shell
is hardened; and the microparticles are isolated. In
the coacervation process, ~U.S. Pat. No. 4,919,929)
which is the most common of the aqueous phase-separation
processes the water-soluble therapeutic agent, which may
be in the form of particles or droplets, is usually
dispersed in an aqueous sol of a hydrophilic colloid
which becomes ionized in water; a second sol of opposite
charge is added; and the mixture is caused to gel by a
dilution with water, an addition of salt, an adjustment
of pH, or a change in temperature, or any combination of
these procedures. Appropriate conditions of coacer-
vation are determined readily by routine trial by those
of ordinary skill in the art because the various usable
polymers differ significantly in physical and chemical
properties according to source and method of isolation
or preparation. A region of coacervation is determined
~y combining solutions or sols of two polymers at
various concentration, temperatures, and levels of pH,
and observing the conditions required for gelation.
From these determinations can be drawn a ternary phase
diagram, showing the area of compatibility and the
region of coacervation, at a given temperature and pH.
The changes in concentration, temperature or pH to
effect gelation are then apparent.
Each preparation of microparticles requires careful
control of conditions, and somewhat different conditions
are required for various material being encapsulated.
The degree of agitation, for example, affects the size
of emulsion droplets. The droplets become smaller in
size with increased agitation. The surface properties
of the droplets may require alterations in the
procedures to insure deposition of matrix material about
the droplets and to minimize formation of particles not
participatins in microencapsulation. The volume of
,2!1~6 0 8 7 8
W094/~738 21 PCT~S94/04~9
water added in the dilution step is not critical, but
generally larger volumes are required to maintain a
stable emulsion when larger droplets are encapsulated.
The above-described phase separation can be adapted
to an alternate technique in which the first step of
forming a stable emulsion or suspension of an nucleic
acids is accomplished by dispersing the nucleic acids in
a solution of the matrix material. Thereafter, the
emulsion is added drop-wise to a non-solvent with
stirring to precipitate the polymer coating material to
form microparticles.
Another type of phase separation technique is the
melt-dispersion microencapsulation technique (see U.S.
Patent No. 4,919,929). A heat-liquefiable, waxy coating
material, preferably of a low-melting wax such as
glycerol distearate, is suspended in an inert liquid
such as a silicone oil or a fluorocarbon in which
neither the wax nor the nucleic acids is appreciably
soluble. The mixture is heated and stirred vigorously
to melt and emulsify the wax. The nucleic acids are
powdered and screened to the desired size range, and the
waxy coating material is dispersed with high shear
agitation. The liquefied wax coats the nucleic acids to
form the waxy liquid-coated microparticles. Thereafter,
the formed microparticles are solidified by continued
agitation which cools the particles. The microparticles
are then isolated by filtration and dried as described
earlier.
Another method of forming the microcapsules is by
interfacial microencapsulation (U.S. Patent No.
4,919,929). This involves bringing two reactants
together zt a reaction interface where polycondensation
of the reactants, usually monomers, occurs to form a
thin, insoluble polymeric film. One technique of
establishing the interface for the encapsulation process
f-2'''~ ~;0878
W094/~738 22 PCT~S94t04~9
is the dispersion or emulsification of the nucleic acids
with one of the reactants which form the condensation
polymer in a continuous phase containing the second
reactants.
Still another method of microencapsulation is by
solvent extraction ~U.S. Patent No.4,915,929). In this
method the desired nucleic acids compound is added to
the polymer matrix material which has been dissolved in
a suitable solvent. The nucleic acids compound may be
soluble or insoluble in the solvent for the polymetric
material. Optionally, the nucleic acids may be
dissolved or dispersed in a second media fluid by adding
it to the polymeric matrix solvent.
The mixture of ingredients in the solvent is
emulsified in a continuous-phase processing medium, the
continuous-phase medium being such that a dispersion of
microdroplets containing the indicated ingredients is
formed in the continuous-phase medium. The continuous-
phase processing medium, commonly water, and the organic
2~ solvent must ~e immiscible. Nonaqueous media, such as
xylene and toluene and synthetic oils and natural oils
can be used as the continuous phase processing medium.
Usually, a surfactant is added to the continuous-phase
processing medium to prevent the microparticles from
2~ agglomerating and to control the size of the solvent
microdroplets in the emulsion. A preferred surfactant-
dispersing medium combination is a 1 to 10 wt %
poly(vinyl alcohol) in water mixture. The dispersion is
formed by mechanical agitation of the mixed materials.
An emulsion can also be formed by adding small drops of
the active agent-wall forming material solution to the
continuous phase processing medium. The temperature
during the formation of the emulsion is not especially
critical but can influence the size and quality of the
_~ microparticles and the solubility of the drug in the
W094/~738 ~ 16 0 8 7 8 PCT~S94/04~9
23
continuous phase. Of course, it is desirable to have as
little of the nucleic acids in the continuous phase as
possible. The temperature must not be so low during
processing as to make too viscous or solidify the
solvent or processing medium. Nor should the
temperature be so high as to evaporate too much medium
or degrade the nucleic acids. Accordingly, the
dispersion process can be conducted at any temperature
which maintains stable operating conditions, preferably
temperature being about 30C to 60C, depending upon the
drug and excipient selected.
The dispersion which is formed by solvent
extraction is a stable emulsion. From this dispersion
the organic solvent immiscible fluid is partially
removed in the first step of the solvent removal
process. The solvent can easily be removed by well-
known techniques such as heating, the application of a
reduced pressure or a combination of both. The
temperature used to evaporate solvent from the
microdroplets is not critical, but should not be so high
that it degrades the nucleic acids, nor should it be so
high as to evaporate solvent at such a rapid rate to
cause defects in the wall forming material. Generally,
from 5 to 75%, and preferably 1 to 25%, of the solvent
is removed in the first solvent removal step.
After the first solvent removal step, the dispersed
microparticles in the solvent immiscible fluid medium
are isolated from the fluid medium by any convenient
means of separation. For example, the fluid can be
decanted from the microparticle or the microparticle
suspension can be filtered. Conventional combinations
of separation techniques can be used if desired.
Following the isolation of the microcapsules from
- the continuous-phase processing medium, the remainder of
3S the solvent in the microcapsules is removed by
W094l~738 2 1 ~ ~ 8 7 8 PCT~S94/~9 ~
24-
extraction. In this second solvent removal step, the
microcapsules can be suspended in the same continuous-
phase processing medium used in the first solvent
removal step, with or without surfactant, or in another
liquid. The extraction medium removes the solvent from
the microcapsules and yet does not dissolve the
microcapsules. During the extraction, the extraction
medium with dissolved solvent must be removed and
replaced with fresh extraction medium. This is best
done on a continual basis. The appropriate rate of
extraction medium replenishment of a given process is
easily determined by one skilled in the art. After the
majority of the solvent has been removed from the
microparticles, the microcapsules are dried by exposure
to air or by other conventional drying techniques, such
as vacuum drying, drying over a desiccant, or the like.
Another method of encapsulation is physical
microencapsulation (U.S. Patent No.4,919,929). Physical
microencapsulation techniques are characterized by the
continuous envelopment of particles or droplets of a
substance in a fluid film, as a melt or solution of the
coating material, in an apparatus containing coaxially-
or sequentially-spaced orifices. Thereafter, the fluid
coating is hardened by a standard cooling technique or
by solvent evaporation.
Among the physical methods for microencapsulation
are those that involve the passage of liquid or solid
core material through a liquid matrix material. The
stream is disrupted by some means to cause the formation
of liquid-coated droplets or particles, and the
resulting particles are cooled or otherwise treated to
solidify the shell material. For example, an aqueous
solution of a substance to be encapsulated is aspirated
into rapidly flowing stream of molten glycerol
3S distearate, and the mixture is ejected through a fine
~ W094/~738 ~;' 216 0 8 7 8 PCT~S94/04~9
nozzle. On emergence from the nozzle, the liquid stream
disintegrates into droplets, each consisting of an
aqueous core surrounded by liquid wax. As these fall
through air, the shells cool and solidify, and
microparticles result. In another version of this
process, the impelling force is supplied by a rotating
member, which ejects the core material centrifugally
through the shell-forming liquid.
The variations of these and other processes of
microencapsulation are many. As is readily apparent to
those skilled in the art, no one process nor any single
set of conditions is applicable to all substances, but
instead a useful process is chosen and the conditions
optimized to achieve the desired results with a specific
nucleic acid. Water soluble nucleic acids were
encapsulated by the phase separation method. Lipid or
organic soluble nucleic acids would be microencapsulated
by the solvent extraction method.
In a preferred method of preparing microparticles
containing a genetic substance, a phase separation
technique is employed whereby a solution of the
polymeric matrix material in a suitable organic solvent
~is prepared. To this solution is added the nucleic
acids suspended or dissolved in water or as fine
particles alone. A non-solvent for the polymeric matrix
material is slowly added to the stirred dispersion
causing the polymeric material to slowly precipitate
around the nucleic acids forming microparticles. The
microparticles are further hardened by the addition of a
second non-solvent for the polymeric matrix material.
The microparticles are then isolated by filtration and
- dried.
The microparticle products of Applicants' invention
are usually made up of particles of a generally
spherical shape, although sometimes the microcapsules
W094/~738 ~16 0 8 7 8 PCTNS94/04~9
26
may be irregularly shaped. The microparticles can vary
in size, ranging from submicron to millimeter diameters.
Preferably, diameters less than 1 to 500 ~m are
desirable for nucleic acids formulations which allows
administration of the microparticles with a standard
gauge needle or other conventional methods. In other
embodiments of the invention, the shaped nucleotide
substance containing matrix material can assume forms
other than microparticles such as rods, wafers,
rectangularly shaped films or blocks. In each case the
nucleic acid substance is distributed throughout the
matrix material. The amount of nucleic acids dispersed
throughout the matrix is an amount sufficient to elicit
the desired therapeutic response as the entrapped
nucleic acid is released by the implanted matrix
material over an extended period of time. These shaped
objects are particularly suitable for subcutaneous
implantation into animals desired to be treated.
The amount of nucleic acids administered to the
animal depends on the particular animal species, target
gene sequence, illness, length of time of treatment, age
of the animal, and amount of treatment desired.
Prior to administration to an animal or group of
animals, the microparticles are suspended in an
acceptable pharmaceutical liquid vehicle, and then the
suspension is injected into the desired portion of the
body of the animal.
The microparticles can be mixed by size or by type
so as to provide for a delivery of nucleic acids to
animals in a multiphasic manner and/or in a manner which
provides different nucleic acids to the animal at
different times, or a mixture of nucleic acids to the
animal at the same time. Other biologically active
agents commonly administered to animals may be blended
with the nucleic acids formulation. For example,
6fo~878
W094/~738 ~ ~ ~ PCT~S94/04~9
antibiotics, antihelmintics, vaccines, or any desired
active agent, either in microparticle form or in
conventional, unencapsulated form may be blended with
the nucleic acids and provided to an animal by the
method of the invention.
MFTHOD-DF.TIVTRY
In a preferred embodiment, the nucleic acids are
administered to humans or animals by a single
administration of the nucleic acid-loaded microcapsule,
such that the microcapsules release the active gene in a
constant or pulsed manner into the animalm, eliminating
the need for repetitive injections. The micro-
encapsulated gene can be injected or implanted or
bombarded directly into the animal. These methods allow
the direct insertion of genes into living animals. In
the preferred method, the microparticle encapsulating
the gene, its promoter and a gold, tungsten, platinum,
ferrite, polystyrene, or latex particle is bombarded
into the tissue. A method of particle bombardment is
disclosed in U.S. Patent No. 4,945,050. The invention,
however, is not limited to particle bombardment.
Delivery of these microparticles can be effected by a
variety of methods including direct injection, receptor
mediated endocytosis, particle bombardment, implants
(subcutaneous or intramuscular) or oral administration.
APP~ICATIONS
The present invention relates to the genetic
transformation of animal, plant and microbial cells as
well as changes in gene expression with the introduction
of new genetic material by transfer of nucleic acid
(synthetic or natural), and slow release biodegradable
polymeric microparticles containing the nucleic acid.
The invention has a wide variety of applications, for
example, in the breeding of plants and animals, the
'' li ;~ 1:
W094/~738 216 0 8 7 ~ PCT~S94/04~9
28
understanding and treatment of diseases, and the
production of protein.
Co-encapsulated compounds containing nucleic acids
can be used for the expression of foreign genes, gene
therapy, and the inhibition of gene activity. Various
examples describing how this technology could be applied
are:
1. ~UMA~ GFNF. T}~F.R~PY
Muscular dystrophy can be treated by implants of
the encapsulated dystrophin gene into the muscle tissue
below the fascia. Uptake of the gene can occur by
transport through the sarcoplasmic reticulum or through
the cut ends of the muscle. Direct in3ection or
bombardment of the encapsulated gene can also allow the
facilitated uptake of the gene into muscle cells.
Knockout of the endogenous defective gene would improve
the effects of the newly expressed gene. Uptake can be
by use of ligands bound to the microencapsulated gene.
2. CANCER T~FATMFNT
Treatment of metastatic cancers can be accomplished
by inserting genes for cytokines or genes for killing
the cancerous cells. Direct insertion of genes into
tumor infiltrating lymphocytes have now been
accomplished (Fitzpatrick-McElligott, Bio/Tech.
10:1036-1040 (1992). An encapsulated gene construct
containing a promoter/regulator enhancer region and
coding region will be inserted into solid tumors. Tumor
infiltrating lymphocyt~s (TIL) cells receiving the
cytokine gene will then circulate to other metastatic
sites. The protein produced intracellularly can then
affect the cancerous cells at this location. After gene
insertion in culture the cells can be grown and
reinfused into the body. The encapsulation process will
serve to protect the gene from degradation.
" ~1~0878
WO 94/23738 PCT/US94tO4239
29
3. TR~NSG~.NIC ~NIMAT. PRODUCTIO~
Animals can be made to express foreign proteins or
altered natural products by introduction of new genetic
information. This method offers a significant advantage
over conventional production of transgenic farm animals.
With this method, a pregnant cow could be injected
in v vo with a gene encoding a recombinant protein,
driven by a ~m~ ry gland-specific promoter. The gene
would be taken up in the developing epithelial cells and
expressed at the time of lactation. This process takes
only a few weeks whereas expression of recombinant
proteins in cows by conventional transgenic technology
takes a minimum of 30 months.
4, (~F.NFTIC I~IUNIZATION
The following example demonstrates a novel method
for inserting genes into living animals (in v vo
studies) in order to express a foreign protein which in
turn would elicit an antigenic response. The
encapsulated gene/promoter sequence for the coat protein
for the HIV virus can be inserted into cells allowing
the production of the antigenic portion of the virus
without the actual virus. This eliminates the risk of
retroviral replication. Several publications now
acknowledge that retroviral vectors pose considerable
risk. Although composed of recombination-incompetent
viruses, these vectors can be reactivated and cause
infection. ~see Temin, Human Gene Thera~y 1~ 123
(1990)) Retroviral vectors have potential problems,
oncogenesis, pathogenesis, and homologous recombination
with helper sequences which can be used to propagate the
vectors. This change can lead to the escape of
competent infectious virus.
- 5. GFNFTIC I~UNIZATIO~
DNA containing the sequence for hepatitis ~ virus
surface antigen driven by the actin promoter is
~ t~-
W094/~738 21~ 0 8 7 8 PCT~S94/04~9
microencapsulated by the phase separation method. 25 mg
of plasmid DNA is encapsulated in 65:35 polylactide and
polyglycolide (PLGA) in 1 g and 2.5 g batches at 5% and
1% loading, respectively. The microspheres preparation
is then injected into the breast muscle of chickens. At
1, 2, 4, and 8 weeks, blood samples are taken and the
titer of anti-HBV antibody determined. The cell
mediated immune response against HBVSA is evaluated by
analysis of lymphokine secretion from chicken WBC
stimulated in the presence of macrophages and antigen.
6. ~FNF KNOCKOUT VIA HOMOT.OGOUS ~FCO~RINATION
A DNA construct encoding a replacement gene for a
defective cellular gene responsible for an inborn error
of metabolism could be engineered for integration via
homologous recombination. The construct could then be
conjugated to a ligand that would target its uptake to
the cell in which the defective gene is expressed. The
conjugate could be microencapsulated and injected into a
patient to provide, over time, enough copies of the new
gene to enter the target cell and knock out the
defective gene, replacing it with the correct sequences.
A possible example of how knockout/replacement therapy
might be of therapeutic use is in hemophilia A.
Hemophilia A is an X-chromosome linked clotting disorder
caused by a defect in the blood clotting factor VIII
gene. A new factor VIII gene might be used to replace a
defective sequence by injection of the microencapsulated
factor VIII gene/asialoglycoprotein conjugate i.p.
There it would be absorbed through the portal
circulation and transported to the liver for
recombination and expression.
F.XAMP T .F~ S
The present invention will now be described by
reference to specific examples which are meant to be
Q 8 7 8
W094t~738 31 PCT~S94/04~9
illustrative only and are not intended to limit the
invention.
Throughout the disclosure the following meanings
are intended: "sec" refers to seconds, "min" refers to
minutes, "h" refers to hours, "d" refers to days, "mL"
refers to milliLiters, and "g" refers to grams.
.F.
MICROF.NC~PSUT.~TION OF
PT.~.~MID DNA IN RIoD~GR~nAR~F~ POT.YY~S
The following three examples make use of plasmid
DNA, and tungsten microcarrier particles microencap-
sulated by the phase separation method. Two plasmids
were used in these experiments; (1) pMH40,
(E. I. du Pont de Nemours and Company, Agricultural
Products, Wilmington, DE) which is 7.3 kilobases (kb) in
length and contains the ~-glucuronidase gene driven by
the cauliflower mosaic virus promoter with an SV-40
virus 3' slice site, and (2) The plasmid pRC/CMV/~-gal,
which is 11.6 kilobases (kb) in length and contains the
~-galactosidase gene and the coding region for neomycin
phosphotransferase II which confers resistance to the
antibiotics kanamycin and G418 (InVitrogen, San Diego,
CA). The gene expression is driven by the
cytomegalovirus promoter. Neither of these plasmids
contain complete viral genomes and neither are
infectious.
For microencapsulation plasmid pMH-40 DNA [500 ~g
or 2000 ~g in 500 ~L 10 mM tris (pH7.4), 1 mM EDTA (TE)
buffer3 or plasmid pRC/CMV/Bgal DNA [50¢ ~g in 500 ~1
10 mM tris (pH 7.4), 1 mM EDTA (TE) buf~er] plus 500 ~l
of 50 mg/mL Herring sperm DNA (Boehringer Mannheim,
Indianapolis, IN) dissolved in TE was incubated in a
- shaking water bath at 65C for 30 min to promote mixing.
The biodegradable polymer used for encapsulation
contained the monomers lactide and glycolide in a ratio
'ti~ 1 6 0 8 7 ~
W094/~738 PCT~S94tO4~9
32
of 65:35 (dl-PLGA) (Medisorb Technologies Int.,
Cincinnati, OH) was weighed into a 5~ mL glass screw-cap
tube and dissolved in 31.7 g of ethyl acetate. To this
was added, 0.75 g of M-17 tungsten microcarrier
particles (Biorad, ~ichmond, CA), the contents were
~igorously agitated and then poured into a 300 mL water
jacketed reaction vessel cooled to 0C. An additional
43.7 g of ethyl acetate was added to the reactor and the
mixture was probe sonicated (Tekmar Model TM375) as 1 mL
of DNA solution was slowly added using a lcc syringe and
18 gauge needle. After 30 sec of sonication, 74 g of
360 fluid 1000 cs silicon oil (Dow Corning, Ithaca, NY)
was added to the reactor over 2 min and this mixture was
then immediately quenched by stirring at room
temperature in 2.5 liters of heptane (Chempure M138
K8JS). After 3.5 h the solid material was collected on
a O.2 ~m filter, washed with heptane and dried in a
vacuum oven for at least 3 d. These microspheres are
extremely sensitive to moisture and to temperatures
above 30C and are therefore stored desiccated at 4C.
Light microscopic pictures (Fig. 1) show the
fluorescently labelled DNA indicated by black arrows can
be microencapsulated. The tungsten core added to the
microparticles are designated by white arrows. Scanning
electron micrographs (Fig. 2) show the size distribution
of the microparticles after encapsulation to range in
size from >1 ~m to <250 ~m. The surface of the
particles appear smooth with the particles sticking to
each other. The action of bombardment and impact
against the screen break up the particles into smaller
components. The results show that D~A can be micro-
encapsulated in a size effective for cellular and tissue
insertion. The dense tungsten core adds the necessary
density for particle bombardment. However, the
invention is not limited to particle bombardment and
~ ~- 2160~7~
W094/~738 33 PCT~S94/04~9
different sizes and shapes can be constructed for direct
injection and implants.
F~MnT~ ~
~FTF~F ~D IDFNTIFICATION
OF PT~SMID DNA ~FTFR MICROFNCAPSUT~TION
This example shows the release of DNA from the
microcapsules over time. The microencapsulated DNA was
recovered by dissolution of 50 mg of the microcapsules
in 500 ~L of 24:1 chloroform/isoamyl alcohol with
simultaneous extraction of the DNA into TE buffer at
room temperature. The DNA in the aqueous phase was
precipitated by the addition of one tenth volume of 3M
sodium acetate (pH 5.2) and 2 volumes of 100% ethanol at
-20C. The integrity of the recovered DNA was analyzed
by ethidium bromide/agarose gel electrophoreis and
visualized by UV illumination. The identity of the
plasmid DNA recovered from the microspheres by this
method was verified by digestion with restriction
endonuclease Eco R1. The plasmid DNA (10 ~g) was cut
overnight at 37C with 20 units of Eco R1. Plasmid
pMH40 was analyzed on a 0.7% agarose gel, stained with
ethidium bromide and photographed. ECO-Rl - cut pMH 40
yield two fragments of the predicted size; 3.3 ~b and
4.0 kb in length. Plasmid pRC/CMV/~-gal has 2 Eco R1
restriction sites and 1 Bam HI site. The plasmid DNA
(10 ~g) was cut overnight at 37C with 50 units of Eco
Rl or Bam HI. After digestion, the cut DNA was analyzed
on a 0.7% agarose gel, stained with ethidium bromide and
photographed. Eco R1-cut DNA yielded two fragments of
the predicted length and Bam HI cut DNA yielded one
restriction fragment.
Controlled release of the DNA in vitro was
- analyzed. 50 mg of Batch 233 microspheres or control
unloaded 65:35 microspheres were suspended in 25 mL of
TE buffer and incubated at 37C in a shaking water bath.
~ 2~6087g
WO 94l23738 PCT/US94/04239
Samples (500 1ll) were taken at 1, 3, 6, 24, 48, and 72
h, pooled precipitated by the addition of one tenth
volume of sodium acetate (pH 5.2) and frozen at -20C.
The precipitated DNA was pelleted ~y centrifugation at
2000 g at 4C for 20 min and the resulting pellet washed
in 70% ETOH at --20C to remove residual salt, and dried
in a DNA Speedvac for 10 min. The pellet was
resuspended in TE and quantitated by W spectrophoto-
metry. DNA recovered from the ~ vitro dissolution
analysis containing pMH40 was analyzed by polymerase
chain reaction. DNA was mixed with 3' and 5' primers,
2.5 units of Amplitaq (Perkin-Elmer, Cetus, Norwalk,
CT), and 2 mM dNTPs in a buffer containing 10 mM tris
(pH 8.3), 50 rnM KC1, 2 mM MgC12, and subjected to 10,
15, 20, and 25 PCR cycles using a DNA thermal cycler
model 480 (Perkin-Elmer Cetus, Norwalk, CT). Each cycle
consisted of 1 min at 94C, 1 min at 42C, and 3 min at
74C. Analysis of the PCR-amplified DNA by agarose gel
electrophoresis and ethidium bromide staining showed
that a single 625 bp DNA fragment was amplified and that
the DNA was released over time (Fig. 3). Thus, these
results indicate that the integrity of the DNA remains
intact during the microencapsulation and release
process.
F~xA~rpT~E 3
GF.NE F.XPP'F.SSION IN pT.~NT CF.T.T.S
~FTF.R DF.T.IVFRY OF F.NCAPSULATFD DNA
Microencapsulated DNA with a tungsten core of .5 ~m
was bombarded into the tissue. The DNA consisted of 75%
herring sperm DNA with 25% B-glucuronidase plasmid
(pMH40). Tungsten/DNA-loaded microspheres (5 mg) were
prepared by suspension in 0.075 mL ice cold 70% ETOH and
sonication on ice. pMH-40 DNA was diluted to 1 ~g/~L
precipitated onto 1.O ~Lm tungsten microcarriers as
35 previously described. Briefly, 60 mg of microcarriers
~ = t ,~
W094/~738 216 0 ~ 7 8 PCT~S94/04~9
were washed in 1 mL of 100% ethanol, sonicated for
30 sec, spun down at 12,000 g and washed with 1 mL of
sterile distilled water, sonicated and spun down again.
The supernatant was decanted and 0.5 mL of sterile
distilled water was added to the microcarriers. Then,
25 ~L of the microcarrier suspension was placed in a new
sterile 1.5 mL microcentrifuge tube and to this was
added 10 ~L of pMH40 plasmid DNA in TE, 25 ~L of 2.5M
CaC12 and 10 ~L of O.lM spermidine (Sigma, St. Louis,
MO) while continuously vortexing. After 10 min of room
temperature incu~ation, the microcarriers with the DNA
precipitated on them were pelleted at 10,000 g for 2 min
and the supernatant removed. The pellet was resuspended
in 0.25 mL 100% ethanol, briefly sonicated, repelleted
at 10,000 g and resuspened in 6C ~L of 100~ ethanol.
Bombardment of cauliflower stems was performed by
spreading 15 ~L of the prepared particles on to a Kapton
macrocarrier disc and allowing the ethanol to evaporate.
Bom~ardment was conducted under 27 in. Hg vacuum and at
2000 p.s.i. rupture disk pressure using the Biolistic
PDS-1000-H~ particle delivery system (BioRad, Hercules,
CA).
~ -glucuronidase (GUS) gene expression was analyzed
in bombarded cauliflower. The tissue was incubated at
room temperature for 18 h under constant illumination
followed by 6 h of darkness and stained for 24 h in the
dark at 37C. The assay solution contained 0.1 nM
Na2HP04 pH7.0, O.S mM K3 Fe~CN]6, 0.5 mM K4 Fe~CN~6 3H2
O, 10 mM Na2 EDTA, 0.5 mg/mL X-glu sodium salt
(Biosynth, Staad, Switzerland3.
The results (Fig. 4~ shows delayed expression of
the transgene activity in cauliflower four days after
~ombardment with microencapsulated DNA. The micro-
capsules contained the ~-glucuronidase marker gene.
Control pieces of tissue without bombardment or after
6~878
W094/~738 PCT~S94/~9
3~
~ombardment without DNA did not show any blue staining.
Thus an exogenous encapsulated gene was delivered to
plant cells and was capable of causing delayed
transformation of the tissue. Th~s, gene expression in
plants was demonstrated by use of the slow release of
the reporter gene B-glucuronidase inserted into intact
cauliflower.
F.~ IP T .F. 4
.~TART.F G~NF ~XPRF.SSION IN ANIMAT.
CFT.TS ~ITH A MIC~OFNC~PSUT.~TFD G~N~
Plasmid pRC/CMV/Bgal and tungsten microcarrier
particles were microencapsulated by the phase separation
method. The conditions fQr mucroen~apsulation were
performed as detailed in Example 1, except for the
following. The concentration of DNA was 500 ~g in
500 ~L of 10 mM tris pH 7.4. The ethyl acetate was
added was at a weight of 44.4 g to the reactor. The
silicon oil 67.9 g instead of 74 g was added to the
reactor. For bombardment, ~C/Lgal DNA was diluted to
1 ~g/~L precipitated onto 1.O ~m tungsten microcarriers
as previously described. Briefly, 60 mg of micro-
carriers were washed in 1 mL of 100% ETOH, sonicated for
30 sec, spun down at 12,00~ g and washed with 1 mL of
sterile distilled water. The supernatant was discarded
and 0.5 mL of sterile distilled water was added. Then,
25 ~L of the microcarrier suspension was placed in a new
1.5 mL microcentrifuge tube and to this was added 10 ~L
of pMH40 plasmid DNA in TE, 25 ~l of 2.5M Cacl and
10 ~L of 0.lM spermidine (Sigma, St. Louis, MO.) while
continuously vortexing. After 10 min at 4C, the
microcarriers with the DN~ precipitated on them were
pelleted at 10,000 g for 2 min and the supernatant
removed. The pellet was resuspended in 0.25 mL 100%
ETOH, briefly sonicated, repelleted at 10,000 g and
3~ resuspended in 60 ~L of 100~ ETOH.
=;, 21~0~78
W094l~738 ` PCT~S94/04~9
Tungsten/DNA-loaded microspheres (5 mg) were
prepared by suspension in 0.075 mL ice cold 70% ETOH and
sonication on ice. Bombardment of confluent CHO cells
was performed by spreading 15 ~L of the prepared
particles on to a Kapton macrocarrier disc and allowing
the ETOH to evaporate. Bombardment was conducted under
15 in. Hg vacuum and at 1350 p.s.i. gas pressure using
the Riolistic PDS-1000-HE particle delivery system
(BioRad, Hercules, CA). After bombardment, the cells
were put back into culture media and grown for two days
under standard conditions without G-418 antibiotics.
After 2 d the cells were selected in media containing
G-418 for 6 to 8 weeks. Live colonies of transformed
cells (Fig. 5) were observed in the bombarded cultures
after 6 weeks of growth in G418 media. Control cultures
bombarded with microcapsules containing only herring
sperm DNA and tungsten showed no live colonies. The
results showed that animal cells in culture can be
stably transformed when microencapsulated genetic
material is inserted into the cells.
~ -galactosidase activity in bombarded and selected
CHO cells was analyzed. Staining for B-galactosidase
enzyme activity was performed by fixation of cells on
Petri plates for 15 min in 0.05~ glutaraldehyde in
phosphate buffered saline (PBS), the fixative was
removed by three rinses with PBS and the cells were
stained by the addition of the X-gal solution and
incubated for 2-~ h. The X-gal solution consisted of
10mM Na P04, 3mM K3 Fe[CN]6, 3mM K4 FelCN]6 3H20, 1 M
MgS04, 1~0mM NaCl, lmM MgCl2, 0.2~ X-gal. Further
evidence for the gene insertion is the blue color seen
after staining for the ~-galactosidase activity
(Fig. 5).
Integration of the ~-galactosidase gene was
determined by PCR analysis of genomic DNA purified from
=
WO 94/23738 2 1 6 ~ 8 7 8 PCT/US94l04239
G-418 resistant CHO cell clones (Fig. 6). Clones
(Fir~. S) were produced by bom~ardment with batch 231
microspheres as described above, dilution and plating in
9Ç well, flat ~ottom tissue culture plates in media
containing G-418 for 3-4 weeks and expanded. DNA was
isolated from the clones usinq the Stratagene DNA
isolation kit. PC~ was performed as above using 2 ~lg of
clone DNA per reaction. Results show that the
B-galactosidase gene has integrated in genomic DNA of
the clones (Fig. 6).
F XA~IP T ~ 5
~NC~PSUT ~TION OF CONJUGATFS AND GENE TRA~SFFR
Ry RFC~PTOR--I~DIATFD ~NDOCYTOSIS
Gene transfer may be accomplished ~y the receptor -
mediated endocytosis pathway, fo- example using
transferrin-polylysine or polylysine-asialoglycoprotein
conjugated to DNA. The strategy of gene transfer by
this method uses bifunctional molecular conjugates
consisting of a cognate moiety for a cell surface
receptor that is linked to a DNA-binding moiety. For
some target cells, however gene transfer by this method
is limited. This limitation is due to degradation ~y
various enzymes, lysosomal, proteases, nucleases. To
enhance DNA protection from enzymatic degradation, we
propose to encapsulate the conjugate and its DNA. One
additional advantage of the proposed encapsulation is
the ability to include, in the microcapsules, selected
lysosomatropic agents which would degrade lysosomes.
Such lysosomatropic agents include chloroquine (Zenke,
et al., Proc. Natl. Acad. Sci. 87:3655-3659 (1990)), and
adenoviruses (Curiel et al., Proc. 2~atl Acad Sci.
88:8850-8854 (1991)) .
To prepare the transferrin-poly(L-Lysine)-DNA
complexes, Applicants would follow the method of Cotten
et al. Proc. ~atl. Ac~d Sci., 87:4033-4037 (1990)) and
W094/~738 PCT~S94/04~9
39
Zenke, et al. Proc. N~tl. Ac~ ~ci , 87:36S5-3659
(1990)). The specific ligation will be accomplished
through modification of the transferrin carbohydrate
moiety. The DNA plasmid pRSVL containing the Photinus
5 pyralis luciferase gene under the control of the Rous
sarcoma ~irus long terminal repeat enhancer/promoter
(DeWet et al. Mol. Cell Riol , 7-:725-737 (1987) will be
used as a reporter gene. Con~ugate-DNA complexes will
be prepared by dilution of 6 ~g of pRSVL DNA in 350 ~L
of HBS (lSOmM NaCl/20 mM Hepes, pH 7.3) followed by
addition to 12 ~g of hTfpL190B diluted in 150 ~L of ~BS.
Complexes will be allowed to form for 30 min at room
temperature. Adenovirus dl312, a replication-
incompetent strain deleted in the Ela region (Jones and
15 Shenk, Proc. NAtl. Ac~d. Sci., 76:3665-3669 (1979)) will
be prepared as described by Curiel et al., (Proc. N~tl.
Acad Sci., 88:8850-8854 (1991)). Chloroquine can be
included in the microcapsules at a concentration of
100 ~M.
Microencapsulation of the DNA-conjugates will be
performed as detailed in Example 1 except for the use of
the plasmid pRSVL - luciferase DNA conjugate at a
concentration of 500 ~g in 500 ~L of lOmM tris pH7.4.
Either adenovirus dl312 or chloroquine (100 ~M) can be
2S included during the encapsulation. Evaluation of the
effect of the microencapsulated DNA-conjugates can be
performed ~n ~ltro on cell lines such as the human
leukemic cell line (Cotten et al., Proc . NAtl. ACA~,
Sci., 87:4033-4037 (1990)) or ~eLa cells grown according
to established conditions (Curiel et al., Proc. NAtl.
Acad. Sci., 88:8850-8854 (1991)). Microencapsulated
DNA conjugates will be added directly to the cells in
culture and incubated at 37C for 48 and 72 h. After
incubation the cells will be harvested for luciferase
3~ gene expression. These results will be compared to
~ 2160878
W094/~738 - PCT~S94/04~9
90,
results on gene expression using DNA-conjugates without
microencapsulation.
Polylysine-asialoglycoprotein conjugated to DNA (Wu
and Wu, (J. R;ol, Ch~m., 263:14621-14624 ~1998)) will
also be used to target liver cells in vivo. The
procedure for conjugation of DNA (pSV2 CAT pl~smid) to
polylysine -asialoglycoprotein is described by Wu and
WU, (J. Ri ol . ~hem ., 263: 14621-14624 (1988) ) . The DNA
conjugate will ~e prepared ~y covalently linking poly-L-
lysine to the galactose-terminal (asialo-)glycoprotein,
asialoorosomucoid (AsOR). Poly-L-Lysine (Sigma) will be
coupled to AsOR in a 2: 1 molar ratio using 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide (Pierce Chem. Co.)
using the procedure of Wu et al. (J. Biol. Chem.,
15 264: 16985-16987 (1989) ) . Complexes with DNA will be
formed at a conjugate to DNA molar ratio 2: 1 as
determined by Wu and Wu, (J. Biol. Chem.,
263 :14621-14624 (1988) ) . Samples will be incubated for
1 h at 25C and then dialyzed for 24 h against 0.15 M
saline through membranes with a molecular weight limit
of 3500 (Spectrum Medical Industries CA.). After
dialysis all samples will be filtered through a 0.2 ~m
membrane (Millipore Corp) to ensure that the complexes
do not contain precipitates.
Encapsulation of the conjugate will be performed as
previously described in Example 1. The molecular
conjugates and the lysosomatropic agents are
encapsulated by the phase separation method in 1 gram of
polylactide and polyglycolide copolymer (PLGA~.
Biodegradable polymer containing a mixture of 65% poly-
lactic and 35% poly-glycolic acids (dl-PLGA) (Medisorb
Technologies Int., Cincinnati, OH) is weighed into a
50 mL glass screw-cap tube and dissolved in 30 g of
ethyl acetate and then poured into a 3G0 mL water
~acketed reaction vessel cooled to 0C. An additional
2~16Q~78
WO 94/23738 41 PCT/US94tO4239
45 g of ethyl acetate is added to the reactOr and the
mixture probe sonicated (Tekmar Model TM375) as 1 mL of
DNA solution containing 25 mg of the conjugate in 10 mM
Tris pH 7.4, 0.1 mM EDTA tTE) buffer is slowly added
using a lcc syringe and 18 gauge needle. After 30 sec
of sonication, 75 g of 360 fluid 1000 cs ~Dow Corning,
Ithaca, NY) silicon oil is added to the reactor over 2
min and this is then immediately quenched by stirring at
room temperature in 2.5 L of heptane ~Chempure M138
~CBJS). After 3.S h, the solid material is collected on
a O.2 ~Lsn filter washed with heptane and dried in a
vacuum oven for at least 3 d. The microcapsules range
in size from greater than 1 ~m to less than 250 llm.
Microspheres are extremely sensitive to moisture and
temperatures above 30C and are stored desiccated at
4C.
In v vo uptake can be measured after injection
intramuscularly or intravenously into Sprague-Dawley
rats with 1 mg of encapsulated pSV2 CAT DNA in a sterile
saline solution. Tissue samples can be monitored for
CAT activity at 24-72 h using the procedure described by
Wu et al. (J. Biol. Chem., 264: 16985-16987 (1989)) .
An improvement in gene expression is predicted from
the use of these procedures because of the protection of
the DNA and conjugate within the microcapsules. Slow
release of the DNA will prolong the effect of the gene
transfer and allow gene expression over an extended
period of time.
F.XA~lP T .F 6
PRODUCTION OF F<F.CO~IRINANT PROTFIN IN CHICEcFN FGG
WITH FNCPPSUT ~T~T~ CONJUGATFT~ DNA
Plasmid DNA is amplified in bacteria, purified and
linearized by enzymatic digestion with restriction
endonuclease. The linear plasmid containing the gene
encoding human growth hormone (hGH), driven by the
W094/~ ~8 216 0 8 7 8 PCT~S94/04~9
42
ovalbumin promoter is conjugated to chicken insulin ~y
cross-linking with disuccinimidyl suberate using the
method of Huckett et al. (8iochem. Pharmacol.,
40:253-263 (1990)). This molecular conjugate is
encapsulated by the phase separation method in 1 g of
polylactide and polyglycolide copolymer (PLGA).
Biodegradable polymer containing a mixture of 65% poly-
lactic and 35% poly-glycolic acids (dl-PLGA) (Medisorb
Technologies Int., Cincinnati, OH) is weighed into a
50 mL glass screw-cap tube and dissolved in 30 g of
ethyl acetate and then poured into a 300 mL water
jacketed reaction vessel cooled to 0C. An additional
45 g of ethyl acetate is added to the reactor and the
mixture probe sonicated (Tekmar Model TM375) as 1 mL of
DNA solution containing 25 mg of the conjugate in 10 mM
Tris pH 7.4, 0.1 mM EDTA (TE) buffer is slowly added
using a lcc syringe and 18 gauge needle After 30 sec
of sonication, 75 grams of 360 fluid 1000 cs silicon oil
(Dow Corning, Ithaca, NY) is added to the reactor over 2
min and this is then immediately quenched by stirring at
room temperature in 2.5 liters of heptane (Chempure M138
KBJS). After 3.5 h, the solid material is collected on
a 0.2 ~m filter washed with heptane and dried in a
vacuum oven for at least 3 d. The microcapsules range
in si2e from greater than 1 ~m to less than 250 ~m.
These microspheres are extremely sensitive to moisture
and temperatures above 30C and are therefore stored
desiccated at 4C.
A bolus of microspheres suspended in CMC injection
vehicle is injected i.p. into 25 week old leghorn laying
hens and the level of growth hormone is determined in
each egg ~y ELISA. Applicants predict that the
conjugated plasmid-insulin complex will be released into
the peritoneal cavity and home to the basal surface of
the oviductal epithelial cells, where it will be taken
WO 94/23738 ~16 0 8 7 8 PCT/US94/04239
43
up by these cells and the hG~ gene will 3~e expressed.
The hormone will be secreted into the oviduct lumen and
incorporated into the chicken egg.
F:X~PT.F. 7
GF.NF~TIC I~SUNI ZATION
The production of an immune response against a
foreign antigen usually requires purification of the
protein, which is then injected into the ~n~ l. The
isolation of enough protein is difficult and time-
consuming. Applicants directly insert encapsulated
genes either ~y injection or particle })ombardment into
skin or muscle. This constitutes a unique method for
vaccination or antibody production.
Innoculation of mice (ICR strain) using the human
growth hormone (hGH) is done using microcapsules
containing the human growth hormone gene under the
transcriptional control of either the human ~-actin
promoter (Leavitt et al., Molec. Cell Biol., 4:1961--1969
(1984)) or the cytomeglovirus (CMV) promoter (Boshart et
al., Cell, 41:521-530 (198S)). The microcapsules for
particle }:ombardment also contain inert particles of
dense material preferably of gold, or tungsten. The
dense particle will improve the momentum and allow
penetration into the cells of the animal. Example 1
describes the method for encapsulation with inert
particles, except that in this example Applicants use
the human growth hormone gene and either the human
~-actin or the CMV promoter. The microcapsules are
propelled by the hand-held Biolistic system (see Tang et
al., Nature, 356:152-154 (1992)). Alternatively, the
bolus of encapsulated microcapsules containing the hGH
gene is injected directly into the muscle of the mice at
several sites.
Production of antibodies directed against hGH is
monitored by assaying sera from tzil-}~leeds for the
W094/~738 ~ 21~ 0 8 7 8 PCT~S94104~9
44
capacity to immunoprecipitate 125I-labelled hGH.
Applicants measure the amount of antibody to hGH by
incubating 1 ~L of sera with 1 ~L l25I-labelled hGH
(DuPont-NEN, 84-88 ~Ci mL~l; 113-116 ~Ci Mg~l) for 1 h at
room temperature. Protein A agarose beads (4 ~L Pierce)
will be added and the slurry incubated for 12-18 h at
4C. The beads will be pelleted by centrifugation and
washed thoroughly with phosphate buffered saline (PBS)
before determining the counts per min (cpm) retained.
Values are normalized to nanograms of hHG precipitated
per ~L of serum. Antibodies against hGH in the sera of
genetically immunized mice are detected by western blot
analysis (see Tang et al., Nature, 356:152-154 (1992)).
The present invention is not to be limited to the
particular embodiment or examples disclosed above, but
embraces all such modified forms thereof as come within
the scope of the following claims.
