Note: Descriptions are shown in the official language in which they were submitted.
WO 94/01139 r PGT/US93/06479
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TARGETING SOMATIC GENE THERAPY TO JOINTS
FIELD OF THE INVENTION
The present invention relates generally to the introduction of
genetic material into the joints and cells comprising the joint
structures for the purpose of somatic gene therapy. Further, it
relates to methods for introducing novel genetic material into cells
within the joint and achieving expression of gene products with
therapeutic potential.
BACKGROUND OF INVENTION
Somatic gene therapy involves the treatment of genetic or
acquired disease by the introduction. of recombinant genes into
somatic cells. Many different organs have been traditionally
considered targets for somatic gene therapy including the bone
marrow, liver, endothelial cells, epithelial cells, fibroblasts, and
muscle. In general, somatic gene therapy has traditionally been
considered for those organs and cells which can be studied in vitro
or ex vivo. One of the reasons for this focus ie that many schemes
for somatic gene therapy involve the harvest of cells from an organ
by surgical means, growing these cells in the laboratory,
introducing genes into these cells using viral vectors, and then
reimplanting these cells into the body using a cellular
tzansplantation procedure. Broad applications of ex vivo schemes
for gene therapy, particularly the transplantation of fibroblasts
into many sites, have been proposed.
More recently attention has turned to schemes for somatic gene
therapy which involve in vivo delivery of recombinant genes to
essential tissues by direct injection or targeted delivery. The
present invention describes a novel approach to somatic gene therapy
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involving the direct delivery of recombinant genes to joint spaces
and cells comprising essential structures of the joint for the
purposes of treating various forms of arthritis or other diseases of
the joints. Major joints may be targeted for somatic gene therapy
by injection of genetic material into the joint space. This therapy
would be intended to prevent the often crippling and painful
manifestations of inflammatory or degenerative arthritis as well as
to enhance the process of repair or regeneration after il~l~ess,
injury, or surgery.
SUMMARY OF INVENTION
An object of the present invention is the transformation of
cells comprising essential structures of the joint.
An additional object of the present invention is a method of
direct delivery of genetic material to the cells comprising
essential structures of the joint.
A further object of the present invention is the in vivo
introduction of genetic material into cells comprising essential
structures of the joint.
Another object of the present invention is a method of
injecting genetic material into the joint space for uptake by cells
constituting structures of the joint.
An additional object of the present invention is the coupling
of genetic materials to non-genetic materials termed vehicles which
enhance the uptake, stability, and expression of genetic material
into cells of the joint.
A further object of the present invention is a method for
treating pathophysiological conditions resulting from inflammatory
processes.
Another object of the present invention is a method for
~ treating hypertrophy or inappropriate proliferation of cellular
elements of the joint.
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A further object of the present invention is a method
for enhancing repair, regeneration, and recovery of essential
structures comprising the joint after surgery or injury.
An additional object of the present invention is a
method for ablating certain cells in the joint.
A further object of the present invention is a method
for generating animal models of arthritis.
In accomplishing the foregoing objects there is
provided in accordance with one aspect of the present invention
a medicament for transfecting a cell in a structure of a joint
comprising (a) a pharmaceutically acceptable carrier, diluent
or vehicle and (b) an effective amount of a DNA vector adapted
to the introduced directly into the joint. The vector is
comprised of elements required for directing transcription of
the genetic material and expression of a specific gene product
encoded by a nucleic acid cassette within the vector. The
following elements are linked sequentially at appropriate
distance for allowing functional expression: a promoter; a 5'
mRNA leader sequence; an initiation site; a nucleic acid
cassette containing the sequence to be expressed; a 3'
untranslated region; and a polyadenylation signal. One or more
of these elements may be eliminated for specific applications.
In an embodiment of this invention the vector is
comprised of DNA sequences which can be inserted into cells of
the joint and can transform these cells to produce novel gene
products.
In a preferred embodiment, the nucleic acid cassette
encodes a protein, polypeptide, or anti-sense RNA. The protein
can be selected from the group including enzymes, proteins
constituents of the extracellular matrix, as well as cytokines,
interleucins, growth factors, toxins, as well as receptors for
CA 02139948 2001-07-26
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these ligands and also natural or genetically modified
receptors for steroid hormones.
In an alternative embodiment, the nucleic acid
cassette encodes a protein, polypeptide, or anti-sense RNA for
the purpose of generating animal models of joint disease. The
protein can be selected from the group including growth
factors, tissue transplantation antigens (histocompatibility
antigens), viral antigens, non-viral antigens, interferon, or
toxins.
The pharmaceutically acceptable carriers, diluents
and vehicles are generally well-known in the art.
In preferred embodiments the medicament contains the
genetic material in conjunction with non-genetic material
termed vehicles which comprise a solutions or suspensions.
This non-genetic material can be selected from the group
including sucrose, protamine, polybrene, spermidine,
polylysine, other polycations, proteins, CaP04 precipitates,
soluble or insoluble particles, or matrices for slow release of
genetic material. The proteins may be selected from the group
including lactoferrin, histone, natural or synthetic DNA
binding proteins, natural or synthetic DNA binding compounds,
viral proteins, non-viral proteins or any combinations of
these. In addition, vehicles may be comprised of synthetic
compounds which bind both to DNA and function as ligands for
receptors on cells comprising structures of the joint.
In specific embodiments of the present invention, the
nucleic acid cassette is transiently expressed by cells of the
joints or is persistently expressed by cells of the joint as an
episome. Alternatively, the nucleic acid cassette is stably
expressed by cells of the joint.
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This approach can be used for the treatment of
pathophysiological conditions resulting from inflammatory
diseases affecting the joints, degenerative diseases of the
joints, proliferative diseases of the joints, repair and
regeneration of essential joint structures as well as other
acquired diseases of the joints. This approach can also be
used to generate animal models of arthritis.
As well known in the art, the medicament may be put
in a commercial package for practical use, transportation,
storage, etc. Such a commercial package usually carriers a
written matter which describes indications of the medicament or
instructions about how to use the medicament.
Other and further objects, features and advantages
will be apparent from the following descriptions of the
presently preferred embodiments in the invention which are
given for the purpose of disclosure and when taken in
conjunction with the accompanying drawings.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows expression of E. cola ~-galactosidase in
synovial structures of the rabbit knee after DNA mediated gene
transfer into the joint. Arraws indicate the regions of synovium
stained with R-gal.
A. Experimental animal after injection with CMV-B-gal.
B. Control animal injected with different DNA construct.
C. Close-up of experimental animal injected with CMV-B-gal.
D. Close-up of control animal.
Figure 2 is a schematic of one proposed mechanism for DNA
mediated gene transfer into synovial cells. Synovial cells form the
boundary between the synovial fluid and adjacent tissues. The
synovial fluid is synthesized by synovial cells and is constantly
remodelled by pinocytosis. DNA injected into the joint can be taken
up during the process of pinocytosis.
Figure 3 is a schematic of one proposed mechanism for DNA
mediated gene transfer into inflammatory cells invading the joint.
Packaging DNA in a form which is preferentially taken up by
phagocytes (such as particulate DNA) can lead to preferential
ingestion, of DNA by phagocytosis. This can be used to alter gene
expression. in phagocytes or ablate these cells.
Figure 4 is a schematic of one proposed mechanism for
targeting DNA to different cells such as chondrocytes and synovial
cells as a complex with a vehicle that is capable of receptor
mediated uptake, membrane fusion, or enhanced pinocytosis.
The drawings are not necessarily to scale and certain features
of the invention may be exaggerated in scale and shoran in schematic
form in the interest of clarity and conciseness.
DETAILED DESCRIPTION OF THE INVENTION
It will be readily apparent to one skilled in the art that
various substitutions and modification may be made to the invention
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disclosed herein without departing from the scope and spirit of the
invention.
The terms "structures comprising the joint" and "cells of the
joint" refer to all of the cellular and non-cellular materials which
comprise the joint and are involved in normal function of the joint
or are present within the joint due to pathological conditions.
These include materials associated with: the joint capsule such as,
synovial membranes, synovial fluid, synovial cells; the
cartilaginous components of the joint such as chondrocytes,
extracellular matrix of cartilage; the bony structures such as bone,
periosteum of bones, periosteal cells, osteoblasts, osteoclasts; the
immunological components such as inflammatory cells, lymphocytes,
mast cells, monocytes, eosinophils; other cells like fibroblasts;
or combinations thereof.
The term "transformed" as used herein refers to transient or
permanent changes in the characteristics (expressed phenotype) of a
cell by the mechanism of gene transfer. Genetic material is
introduced into a cell in a form where it expresses a specific gene
product or alters the expression or effect of endogenous gene
products..
The term "transfection" as used herein refers to the process
of introducing a DNA expression vector into a cell. Various methods
of transfection are possible including microinjection, CaP04
precipitation, liposome fusion (e. g. lipofection) or use of a gene
gun.
The term "transient" as used herein relates to the
introduction of genetic material into a cell to express specific
proteins, peptides, or RNA etc. The introduced genetic material is
not integrated into the host cell genome or replicated and is
accordingly eliminated from the cell over a period of time.
The term "stable" as used herein refers to the introduction of
genetic material into the chromosome of the targeted cell where it
integrates and becomes a permanent component of the genetic material
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in that cell. Gene expression after stable transduction can
permanently alter the characteristics of the cell leading to stable
. transformation.
The term "persistent" as used herein refers to the
introduction of genes into the cell together with genetic elements
which enable episomal (extrachromosomal) replication. This can lead
to apparently stable transformation of the characteristics of the
cell without the integration of the novel genetic material into the
chromosome of the host cell.
The term "ligand" refers to a protein capable of binding to a
receptor on a cell.
The term "nucleic acid cassette" as used herein refers to the
genetic material of interest which can express a protein, or a
peptide, or RNA to incorporate it transiently, permanently or
episomally into the structures comprising the joint. The nucleic
acid cassette is positionally and sequentially oriented in a vector
with other necessary elements such that the nucleic acid in the
cassette can be transcribed and, when necessary, translated in the
' cells of the joint.
The term "vector " as used herein refers to a construction
comprised of genetic material designed to direct transformation of
a targeted cell. A vector contains multiple genetic elements
positionally and sequentially oriented with other necessary elements
such that the nucleic acid in a nucleic acid cassette can be
transcribed and when necessary translated in the transfected cells.
In the present invention the preferred vector comprises the
following elements linked sequentially at appropriate distance for
allowing functional expression: a promoter; a S' mRNA leader
sequence; an initiation site; a nucleic acid cassette, containing
3'0 the sequence to be expressed; a 3' untranslated region; and a
polyadenylation signal.
The term "genetic material" as used herein refers to
contiguous fragments of DNA or RNA. The genetic material which is
WO 94/01139 PCT/US93106479
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introduced into cells comprising essential structures of the joint
according to the methods described herein can by any DNA or RNA.
For example the nucleic acid can be: 1) normally found in cells of
the joints, 2) normally found in cells of the joints but not
expressed at physiologically appropriate levels, 3) normally found
in cells of the joints but not expressed at optimal levels in
certain pathological conditions, 4) novel fragments of genes
normally expressed or not expressed in structures of the joint, 5)
synthetic modifications of genes expressed or not expressed within
structures the joints, 6) any other DNA which may be modified for
expression in cells of the joint, and 7) any combination of the
above.
The term "pharmacological dose" as used herein refers to a
dose of vector and level of gene expression resulting from the
action of the promotor on the nucleic acid cassette when introduced
into the appropriate cell type which will produce sufficient
protein, polypeptide, or antisense RNA to either (1) increase the
level of protein production, (2) decrease or stop the production of
~ a protein, (3) inhibit the action of a protein, (4) inhibit
proliferation or accumulation of specific cell types, (5) induce
proliferation or accumulation of specific cell types. As an
example, if a protein is being produced which causes the
accumulation of inflammatory cells within the joint, the expression
of this protein can be inhibited, or the action of this protein be
interfered by expression of appropriate regulatory proteins. The
dose will depend on the protein being expressed, the promoter,
uptake and action of the protein RNA. Given any set of parameters,
one skilled in the art will be able to determine the dose.
The term "ablation agent" as used herein refers to an agent
which is capable of destroying the cell in which it is present.
Examples of ablation agents used in the present invention include
diphtheria toxin and herpes thymidine kinase.
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The term "vehicle" as used herein refers to non-genetic
material combined with the vector in a solution or suspension which
enhance the uptake, stability, and expression of genetic material
into cells of the~joint. Examples of a vehicle include: sucrose,
S protamine, polybrene, spermidine, polylysine, other polycations,
proteins, CaP04 precipitates, soluble and insoluble particles, or
matrices for slow release of genetic material. The proteins may be
selected from the group including lactoferrin, histone, natural or
synthetic DNA binding proteins, natural or synthetic DNA binding
compounds, viral proteins, non-viral proteins or any combinations of
these. In addition, vehicles may be comprised of synthetic
compounds which bind both to DNA and function as ligands for
receptors on cells comprising structures of the joint.
Examples of proteins or polypeptides which can be expressed by
the vector in the transformed cells of the joint for the purposes of
somatic gene therapy include enzymes, extracellular matrix elements,
cytokines, interleucins, growth factors, toxins, as well as
receptors for these ligands and also natural or genetically modified
receptors for steroid hormones.
Examples of proteins or polypeptides which can be expressed by
the vector in the transformed cells of the joint for the purposes of
generating animal models of arthritis include growth factors, tissue
transplantation antigens (histocompatibility antigens), viral
antigens, non-viral antigens, interferon, or fragments of diphtheria
toxin.
One specific embodiment of the present invention ie a method
of transfecting a cell in a structure of a joint comprising the step
of introducing a DNA vector directly into the j oint, said vector
comprised of the following elements linked sequentially at
appropriate distances for allowing functional gene expression: a
promotor; a 5' mRNA leader sequence, an initiation site, a nucleic
acid cassette containing the nucleic acid sequence to be expressed,
a 3' untranslated region, and a polyadenylation signal. One skilled
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in the art will readily recognize how to order and position these
elements such that the nucleic acid sequence is expressed. Further,
one skilled in the art will readily recognize that certain elements
of this DNA vector may be eliminated for specific applications. In
preferred embodiments, the DNA vector without the nucleic acid
cassette will contain a restriction site for insertion of the
specific cassette. Once the specific cassette is inserted, the
restriction site may no longer be present.
In the present invention the nucleic acid cassette is
activated with a promotor. The promotor is any of the wide variety
of promotors known in the art. Examples of the promotors which are
used in the present invention are the retroviral LTR promotor,
cytomegalovirus promotor, dihydrofolate reductase promotor, viral
promotors, or non-viral promotors. Alternatively, the promotor can
be selected from those shaven to specifically express in the select
cell types which may be found associated with the structures of the
joint such as synovial cells, fibroblasts, lymphocytes, periosteal
cells, chondrocytes, osteoblasts, osteoclasts, or more than one of
these cell types.
One skilled in the art will recognize that the selection of
the promotor will depend on the vector, the nucleic acid cassette,
the cell type to be targeted, and the desired biological effect.
One skilled in the art will also recognize that in the selection of
a promotor the parameters can include: achieving sufficiently high
levels of gene expression to achieve a physiological effect,
maintaining a critical level of gene expression, achieving temporal
regulation of gene expression, achieving cell type specific
expression, achieving pharmacological, endocrine, paracrine, or
autocrine regulation of gene expression, and preventing
inappropriate or undesirable levels of expression. Any given set of
selection requirements will depend on the conditions but can be
readily determined once the specific requirements are determined.
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Those promotors which naturally occur in the cells comprising
structures of the joint will be preferred.
Introduction of a vector into cells comprising structures of
the joint can be used to treat a variety of pathological conditions.
These conditions can result from the abnormal production of a
protein or a polypeptide, for example too little or too much of a
protein or polypeptide, the production of an abnormal protein or
polypeptide, atopic production of a protein or peptide, or abnormal
regulation of production of a protein or polypeptide. Introduction
of a vector into cells comprising structures of the joint can also
be used to provide proteins or peptides or genetic elements (DNA or
RNA) with therapeutic actions. One skilled in the. art will
recognize that even complex pathophysiological events such as
inflammatory diseases, degenerative, diseases, injury and
regeneration can be understood as comprising a series of molecular
interactions between proteins and can be treated according to the
embodiment of this invention.
For example, in the treatment of a pathological condition the
vector in a vehicle will be introduced into cells comprising
structures of the joint by injecting a pharmacological dose of the
vector and vehicle into a joint.
The preferred embodiments of this invention involves transient
or persistent expression within the joint. This is preferable to
stable expression since it enables adjustment of the level of
expression in response to the evolution of the disease process.
Stable expression may presently be achieved by the use of
viral vectors or the transplantation of cells which are stably
transformed with viral or DNA vectors.
Specific diseases which can be treated by administration of
vectors to cells within the joint include various arthritis,
avascular necrosis, or injuries requiring repair and regeneration of
structures comprising the joint. Various types of arthritis can be
treated. A list of these various types are shown below. This list
~~.~99~~3
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is based on a classification of joint disorders: (after table 358-1,
p. 2048 Harrison's Principles of Internal Medicine, Thorn, G. W.,
Adams, R. D., Braunwald, E., Isselbacher, R. J., Petersdord, R. G.,
McGraw HillBook Company, 1977)
I. Periarticular
A. Tendinitis
B. Bursitis
C. Fibrositis
D. Bone lesions
E. Soft Tissue inflammation
II. Articular
A. Cartilage and ligaments
1. Degenerative joint disease
2. Traumatic disorders
3. Neuropathic arthropathy
4. Metabolic disorders
B. Synovium
1. synovial tumors
2. pigmented villonodular synovitis
3. hemorrhagic disorders
4. septic disorders
5. crystal induced disorders (gout)
a. Immune complex disease and vasculitis
b. Systemic lupus erythematosis
c. rheumatoid arthritis
d. Reiter's syndrome
e. Psoriasis
f. ankylosing spondylitis
g. scleroderma
h. arthritis of intestinal disease
The method of treating a pathophysiological condition,
or
repairing and regenerating structures of the joint comprises
the
steps of
injecting
a joint
of a human
with a
pharmacological
dose of
21.399$ ,
WO 94/01139 PCT/US93/06479
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a DNA vector in a vehicle, wherein the nucleic acid cassette in the
vector encodes a protein, polypeptide or RNA. The vector is taken
up by appropriate cells within the joint and expresses the protein,
polypeptide or RNA. Specific embodiments of the invention include
treatment of disorders listed above.
Specific embodiments of the present invention include a method
of ablation of inflammatory cells in a joint comprising the step of
introducing a vector into the joint, said vector comprised of the
following elements linked sequentially at appropriate distances for
allowing function expression: a promoter which is specifically
expressed in leucocytes; a 5' mRNA leader sequence; an initiation
site; a nucleic acid cassette containing the sequence for an
ablation agent; a 3' untranslated region; and a polyadenylation
signal; wherein the vector is targeted for selective uptake by
phagocytic cells in the joint. In the preferred embodiment, the
vector is directly targeted by attaching the vector to a molecule
which attaches to the surface of the phagocytic cell.
In specific embodiments of the present invention for the
treatment of arthritis, a soluble receptor for cytokines can be
used. Examples of a soluble receptor include IL-1 or IL-6.
An alternative embodiment of the present invention includes a
method of treating arthritis in humans comprising the step of
injecting an inflamed joint of a human with a pharmacological dose
of a DNA vector in a vehicle, wherein the nucleic acid cassette in
the vector encodes a sequence for a steroid receptor. This can be
the normal receptor or it can be a genetically modified receptor.
When treating pathophysiological conditions or repairing or
regenerating structures of the joint, it is found that a useful
nucleic acid cassette encodes antisense RNA to prostaglandin
synthase.
Another embodiment of the prevent invention is a method of
making an animal model for inflammatory arthritis comprising the
step of injecting the joint of an animal with a functional DNA
PCT/US93/06479
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vector in a vehicle. The nucleic acid cassette in the vector
encodes a~transplantation antigen, cell adhesion antigen, antigen
from micro-organisms, viral antigen, synthetic antigen or
recombinant antigen. Examples of the transplantation antigen
include the histocompatibility antigens, Class I transplantation
antigen, Class II transplantation antigen and other transplantation
antigens, allogeneic transplantation antigen and xenogeneic
transplantation antigen.
Another embodiment of the present invention is a transformed
synovial cell comprised of nucleic acid incorporated into a synovial
cell by gene transfer. In,specific embodiments, the nucleic acid
can be any of the genetic materials described above. Synovial cells
are capable of expressing either protein, polypeptide or antisense
RNA. Generally, synovial cells can express cytokines, interleucins,
p g ~ eneticall modified rece tots for
rece tots for natural li ands, g y P
natural ligands, inhibitors of natural ligands, steroid receptors,
genetically modified steroid receptors, cell adhesion molecules,
genetically modified adhesion molecules, enzymes affecting
prostaglandin metabolism, enzymes involved in extracellular matrix,
RNA molecules inhibiting production of transplantation antigens,
cell adhesion molecules, receptors, or enzymes involved in
prostaglandin metabolism. The gene in the transformed synovial
cells can specifically encode a steroid receptor, IL-1, IL-6, IL-8,
or soluble IL-1 receptor, the transplantation antigen or an
antisense RNA to prostaglandin synthase.
Another embodiment of the present invention is a method of
targeting a DNA vector to joints comprising the steps of identifying
human antibodies from patients with sutoimmune arthritis, cloning
the antibodies to develop monoclonals With the same epitope binding
determinants and coupling DNA vectors to the monoclonals; wherein
the DNA/monoclonal complex is delivered to the cells of the joint.
In the preferred embodiment the identified antibodies have the
property of binding specifically to proteins on cells within the
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joint. One skilled in the art recognizes that a variety of methods
are available for identifying antibodies.
An alternative method of gene therapy is a method of
introducing DNA into an extracellular space enclosed by a cellular
membrane. For example, this method of gene therapy can be used in
both humans and animals and it comprises the step of introducing a
pharmacological dose of a DNA vector in a vehicle ints,o an
extracellular space in the human or animal. The extracellular space
is enclosed by a cellular membrane. An example of this type of
treatment would be the introduction of a DNA vector into the
synovium of the joint.
Another method for gene therapy in humans and animals is
introduction of a pharmacological dose of DNA vector in a vehicle
into a fluid space in the animal or human. This fluid space is
reabsorbed or remodelled by surrounding cells. Examples of this
type of gene therapy would be the introduction of a DNA vector into
a follicle of the thyroid, the synovium of the joint or the vitreous
of the eye.
Another embodiment of the present Invention is a method for
gene therapy in humans and animals, comprising the step of
introducing a pharmacological dose of a DNA vector into a fluid of
the animals or humans . The f low and removal of this f luid is by
endocytosis or pinocytosis from the surrounding cells. Examples of
this occurs in the thyroid follicles, synovium of the joint, and in
the vitreous of the eye.
Another embodiment of the present invention is a method of
gene therapy in animals or humans comprising the step of introducing
a pharmacological dose of DNA vector in a vehicle Into a fluid space
of the animal or human. The flow and removal of the fluid through
a membrane causes the fluid to be filtered and the cells of the
membrane then uptake the filtered DNA. An example of this is in the
kidney and in the central nervous system, including the spinal
column and brain.
. , ., ,
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One skilled in the art will readily recognize that a variety
of other tissues, fluid space and extracellular spaces may be used.
As long as they meet the specific criteria of being surrounded by a
cellular membrane or the uptake in removal of the fluid or the
S filtering of the fluid.
The following examples are offered by way of illustration and
are not intended to limit the invention in any manner.
Example 1
Introduction of a Marker Gene into Cells of the Joint
A vector comprising the cytomegalovirus immediate early
promotor, S' untranslated sequences and an intron from SV40, 3'
untranslated sequences from SV40 including the polyadenylation
signal and a nucleic acid cassette containing the E. cola beta-
galactosidase gene was combined with a vehicle containing 20x
sucrose at pH 7.4. This solution was injected into the knee joint
of rabbits using a transpatellar injection. As a control, an
identical vector containing the E. coli chloramphenicol
acetyltransferase gene in place of the beta-galactosidase gene was
injected into joints on the opposite leg using identical methods.
Three days after injection animals were sacrificed, the joint was
disarticulated, and stained in a solution containing X-gal at pH
7.2. Under these conditions cells taking up and expressing the E.
cola beta-galactosidase gene will be stained blue. Cells not
expressing beta-galactosidase do not stain with this dye under these
conditions.
The analysis showed that there was diffuse blue staining in
regions of the joint representing the synovial cells of joint
injected with the beta-galactosidase containing vector (figures lA,
1C). No staining was observed over the tendons of bony surfaces of
the joints. No blue staining was apparent in joints injected with
the control vector (figures 1B, 1D).
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These results demonstrate that cells of the joint are capable
of taking up DNA vectors and are capable of expressing gene products
encoded by the vector.
Example 2
l~xpression of anti-inflammatory factors
to prevent inflammatory arthritis
The inflammatory attack on joints in animal models and human
diseases may be mediated, in part, by secretion of cytokines such as
IL-1 and IL-6 which stimulate the local inflammatory response. The
inflammatory reaction may be modified by local secretion of soluble
fragments of the receptors for these ligands. The complex between
the ligand and the soluble receptor prevents the ligand from binding
to the receptor which is normally resident on the surface of cells,
thus preventing the stimulation of the inflammatory effect. Therapy
consists of the construction of a vector containing the soluble form
of receptors for appropriate cytokines (for example I1-1 or IL-6)
together with promotors capable of inducing high level expression in
structures of the joint and a vehicle which enables efficient uptake
of this vector. This DNA is injected into affected joints where the
secretion of an inhibitor for IL-1 such as a soluble IL-1 receptor
or natural IL-1 inhibitor modifies the local inflammatory response
and resulting arthritis.
This method is useful in treating episodes of arthritis which
characterize many "autoimmune" or "collagen vascular" diseases.
This method can also prevent disabling injury of large joints by
inflammatory arthritis.
Example 3
Induction of "steroid response" b~,ggne transfer of steroid
receQtors into cells of the joint
Current therapy for severe arthritis involves the
administration of pharmacological agents including steroids to
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depress the inflammatory response. Steroids can be administered
systemically or locally by direct injection into the joint space.
Steroids normally function by binding to receptors within the
cytoplasm of cells. Formation of the steroid-receptor complex
changes the structure of the receptor so that it becomes capable of
binding to specific sequences within the genome of the cell and
altering the expression of specific genes. Genetic modifications of
the steroid receptor can be made which enable this receptor to bind
naturally occurring steroids with higher affinity, bind
pharmacological forms of steroid at higher affinity, or bind to
synthetic steroids. Other modifications can be made to create
steroid receptor which is "constitutively active" meaning that it is
capable of binding to DNA and regulating gene expression in the
absence of steroid altogether.
One approach to treating arthritis is to introduce a vector in
which the nucleic acid cassette expresses a genetically modified
steroid receptor into cells of the joint. Expression of a
constitutively active steroid receptor within cells of the joint
induces the therapeutic effects of steroids without the profound
systemic toxicity of these drugs. Of particular importance is the
ability to target these genes differentially to specific cell types
(for example synovial cells versus lymphocytes) to affect the
activity of these cells.
Alternatively, steroid receptors which have a higher affinity
for natural steroids can be introduced into the joint. These
receptors exert an increased anti-inflammatory effect when
stimulated by physiological concentrations of steroids or lower
doses of pharmacologically administered steroids. Alternatively,
constitution of a steroid receptor which binds a novel steroid
enables the use of drugs which would affect only cells taking up
this receptor. These strategies obtain a therapeutic effect from
steroids on arthritis without the profound systemic complications
associated with these drugs.
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Example 4
Inhibition of prostaglandin spnthase
Drugs which inhibit the enzyme prostaglandin synthase are
important agents in the treatment of arthritis. This is due, in
part, to the important role of certain prostaglandin in stimulating
the local immune response. Salicylates are widely used drugs but
can be administered in limited doses which are often inadequate for
severe forms of arthritis.
Gene transfer is used to inhibit the action of prostaglandin
synthase specifically in affected joints by the expression of an
antisense RNA for prostaglandin synthase. The complex formed
between the antisense RNA and mRNA for prostaglandin synthase
interferes with the proper processing and translation of this mRNA
and lowers the levels of this enzyme in treated cells.
Alternatively, genes encoding enzymes which alter
prostaglandin metabolism can be transferred into the joint. These
have an important anti-inflammatory effect~by altering the chemical
composition or concentration of inflammatory prostaglandin.
Example 5
Generatin~P an animal model of inflammator9 arthritis
Inflammatory arthritis is thought to result from an sutoimmune
attack on cells comprising essential structures of the joint. The
association of various forms of arthritis with certain tissue
transplantation (Histocompatibility) antigens, suggests that these
antigens, or dysregulation of these antigens, may play an important
role in the genesis of these diseases.
Animal models of inflammatory arthritis can be generated by
gene transfer of vectors capable of expressing heterogenic (not
self) or xenogeneic (other species) transplantation antigens within
the joint, thus mimicking the effect of dysregulation of tissue
transplantation antigens. This induces an sutoimmune attack not
only on the transplantation antigens themselves, but on other
WO 94/01139 PCT/US93/06479
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antigens within the joint which are presented to the immune system,
in an abnormal manner.
Alternatively, overproduction of interferon in tissues is
thought to induce dysregulation of class I antigens and potentiate
a inflammatory reaction. This can be modeled in animals by
introduction of vectors expressing interferon into cells of the
j oint .
Example 6
Gene transfer to enhance revair or rer~eneration of ioints
The regenerative capacity of the joint is limited by the fact
that chondrocytes are not capable of remodelling and repairing
cartilaginous tissues such as tendons and cartilage. Further,
collagen which is produced in response to injury is of a different
type -- lacking the tensile strength of normal collagen. Further,
the injury collagen is not remodeled effectively by available
collagenase.
Gene transfer using promoters specific to chondrocytes (i.e.,
collagen promoters) is used to express different collagens or
collagenase for the purpose of improving the restoration of function
in the joints and prevent scar forsaation. Gene transfer into
fibroblasts or muscle cells in the environment of the joint is used
to locally secrete growth factors such as IGF-1. The growth factors
maintain muscle mass and proliferation and enhance the strength of
the damaged joint.
Gene transfer for these purposes is affected by direct
introduction of DNA into the joint space where it comes into contact
with chondrocytes and synovial cells. further, the genes infiltrate
into the environment of the joint where they are taken up by
fibroblasts, myoblasts, and other constituents of periarticular
tissue. Additionally, the gene gun can be used interoperatively or
via arthroscopy.
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Example 7
Treatment of outv arthritis by gene transfer
Gout is caused by the accumulation of uric acid in joints.
This remains a common and painful disorder in the aging population
despite medical management. Gene transfer into the joints ie used
to express products (for example enzpanes) capable of degrading uric
acid to non-toxic products. These products include urease or urate
oxidase which are capable of metabolizing uric acid or urate binding
globulins which render this compound more soluble and thus prevent
crystalline formation within the joint.
Example 8
Persistent expression using episomal vectors
In each of the foregoing examples, transient expression of
recombinant genes induces the desired biological response. In some
diseases more persistent expression of recombinant genes is
desirable. This is achieved by adding elements which enable
extrachromosomal (episomal) replication of DNA to the structure of
the vector. Vectors capable of episomal replication are maintained
as extrachromosomal material and can replicate. These sequences
will not be eliminated by simple degradation but will continue to be
copied. Episomal vectors provide prolonged or persistent
expression, though not necessarily stable or permanent, expression
of recombinant genes in the joint. Persistent as opposed to stable
expression is desirable to enable adjustments in the pharmacological
dose of the recombinant gene product as the disease evolves over
time.
Example 9
Gene delivery usin~~ the-gene gun_
The repair and regeneration of tissues comprising the joint
following injury or surgery is enhanced by directly delivering
recombinant genes to specific sites at the time of surgery. This is
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done in conjunction with surgical reconstructions or arthroscopy
using the "gene gun" (Yang, N.S. et al.; In vivo and in vitro gene
transfer to mammalian somatic cells by particle bombardment; Proc.
Natl. Acad. Sci. USA 87:9568-72 (1990)) to deliver particles coated
with DNA to specific regions of the joint. The use of the gene gun
and particle bound DNA enables constitution of gene expression at
highly specific locations.
Example 10
Vehicles for r~ene delivery into cells of the ioint
Initial experiments used DNA in solution for gene transfer
into cells of the joint. This DNA is taken up by synovial cells
during the process of these cells continually resorbing and
remodeling the synovial fluid by secretion and pinocytosis. Gene
delivery is enhanced by packaging DNA into lipophilic particles (for
example lipofectin) which binds nonspecifically to hydrophobic
membranes resulting in a fusion of the particle with the membrane
and release of the DNA into the cytoplasm. Similarly, complexes of
DNA and proteins which bind to receptors on the surface of cells in
the joint enhances uptake and expression. Alternatively,
particulate DNA complexed with CaP04 or polycations can be efficient
substrates for phagocytosis by monocytes or other inflammatory
cells.
Example 11
Ablation of inflammatory cells invadinr~ the ioint.
Inflammatory cells invading the jo2nt are ablated using
vectors which contain the diphtheria toxin gene under the control of
a promotor which is expressed only in leukocytes. These vectors are
delivered to the joint as particles and are selectively taken up by
phagocytotic cells. The uptake and expression of this vector
results in expression of diphtheria toxin which is lethal to that
specific cell. Alternatively, a vector which expressed herpes
2139948
- 23 -
thymidine kinase gene under the control of a specific promoter
could be introduced into cells, and these cells are then
ablated by administration of acytovin.
All patents and publications mentioned in this
specification are indicative of the levels of those skilled in
the art to which the invention pertains.
One skilled in the art will readily appreciate that
the present invention is well adapted to carry out the objects
and obtain the ends and advantages mentioned, as well as those
inherent therein. The present examples of DNA vectors along
with the methods, procedures, treatments, molecules, and
specific compounds described herein are presently
representative of preferred embodiments, are exemplary, and
are not intended as limitations on the scope of the invention.
Changes therein and other uses will occur to those skilled in
the art which are encompassed within the spirit of the
invention as defined by the scope of the claims.
60724-2257
s _i;
