Note: Descriptions are shown in the official language in which they were submitted.
WO 94/20517 PCT/US94/02414
2157782
GBN13: TR~N~FB~ FOR q'R~:ATING A CON~-~v~; TI881JE
OF A MA~'`r-T~N HO8T
~ROSS-~EF~RENCE TO p~F~T.~TF~n APPLICATION
This is a continuation-in-part application of United
States Application Serial No. 07/630,981, filed Decem~er 20,
1990, now r~n~ing.
sAcKGRouND OF 'rR~ lN V~llON
Field Of The Invent;on
The present invention relates to a method of
i~.LLoducing at least one gene ~nco~;ng a product into at least
one cell of a connective tissue of a mammalian host for use in
treating the mammalian host. This method discloses employing
DNA vector molecules cont~ining a gene encoding the product
and infecting the conn~çtive tissue cells of the mammalian
host using the DNA vector molecule. This invention provides
a method of introducing at least one gene ~nco~ing a product
into at least one cell of a connective tissue of a mammalian
host for use in treating the mammalian hcst including
employing non-viral means for effecting such introduction.
The present invention also relates to a method to
produce an animal model for the study of connective tissue
pathology.
The present invention further relates to a method of
using a gene ~nco~;ng a truncated interleukin-l receptor to
resist the deleterious pathological changes associated with
arthritis. More specifically, this invention provides a
method wherein a gene coding for an extracellular interleukin-
1 binding domain of an interleukin-l receptor is introduced
into synovial cells of a mammalian host in vivo for
neutralizing the destructive activity of interleukin-l upon
cartilage and other soft tissues. As an alternative, the
patients own synovial cells are transduced in vitro and
WO94/20517 PCT~S94/0~14
.
introduced back into the affected joint, using transplantation
procedures such as for example, intra-articular injection.
As an alternative to the in vitro manipulation of
synovia, the gene encoding the product of interest is
introduced into liposomes and injected directly into the area
of the joint, where the liposomes fuse with synovial cells,
resulting in an in vivo gene transfer to synovial tissue. As
an additional alternative ~o the n vitro manipulation of
synovia, the gene encoding the product of interest is
introduced into the area of the joint as naked DNA. The naked
DNA enters the synovial cell, resulting in an in vivo gene
transfer to synovial tissue.
As an another alternative, hematopoietic progenitor cells
or the mature lymphoid or myeloid cells may be transfected
n vitro, recovered and injested into the bone marrow of the
patient using tec-hniques known to the skilled artisan.
Brief Description Of The Related Art
Arthritis involves inflammation of a joint that is
usually accompanied by pain and frequently changes in struc-
ture. Arthritis may result from or be associated with a
number of conditions including infection, immunological
disturbances, trauma and degenerative joint diseases such as,
for example, osteoarthritis. The biochemistry of cartilage
degradation in joints and cellular changes have received
considerable investigation.
In a healthy joint, cells in cartilage
(chondrocytes) and the surrounding synovium (synoviocytes) are
in a resting state. In this resting state, these cells
secrete basal levels of prostaglandin E2 and various neutral
proteinases, such as, for example, collagenase, gelatinase and
stromelysin, with the ability to degrade cartilage. During
the development of an arthritic condition, these cells become
activated. In the activated state, synoviocytes and
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21S7782
chondrocytes synthesize and secrete large amounts of
prostaglandin E2 and neutral proteinases.
In efforts to identify pathophysiologically relevant
cell activators, it has been known that the cytokine
interleukin-1 activates chondrocytes and synoviocytes and
induces cartilage breakdown n vitro and in vivo.
Additionally, interleukin-1 is a growth factor for
synoviocytes and promotes their synthesis of matrix, two
properties suggesting the involvement of interleukin-1 in the
synovial hypertrophy that accompanies arthritis. In contrast,
interleukin-1 inhibits cartilaginous matrix synthesis by
chondrocytes, thereby suppressing repair of cartilage.
Interleukin-1 also induces bone resorption and thus may
account for the loss of bone density seen in rheumatoid
arthritis. Interleukin-1 is inflammatory, serves as a growth
factor for lymphocytes, is a chemotactic factor and a possible
activator of polymorphonuclear leukocytes (PMNs). When
present in a sufficient concentration, interleukin-1 may cause
fever, muscle wasting and sleepiness.
The major source of interleukin-1 in the joint is
the synovium. Interleukin-1 is secreted by the resident
synoviocytes, which are joined under inflammatory conditions
by macrophages and other white blood cells.
Much attention has been devoted to the development
of a class of agents identified as the "Non-Steroidal Anti-
Inflammatory Drugs" (hereinafter "NSAIDs"). The NSAIDs
inhibit cartilage synthesis and repair and control inflamma-
tion. The mechanism of action of the NSAIDs appears to be
associated principally with the inhibition of prostaglandin
synthesis in body tissues. Most of this development has
involved the synthesis of better inhibitors of cyclo-
oxygenase, a key enzyme that catalyzes the formation of
prostaglandin precursors (endoperoxides) from arachidonic
acid. The anti-inflammatory eEfect of the NSAIDs is thought
WO94/20~17 PCT~S94/0~14
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to be due in part to inhibition of prostaglandin synthesis and
release during inflammation. Prostagl~n~;n~ are also believed
to play a role in modulating the rate and extent of leukocyte
infiltration during inflammation. The NSAIDs include, such
as, for example, acetylsalicylic acid (aspirin), fenoprofen
calcium (Nalfon~ Pulvules~, Dista Products Company), ibuprofen
(Motrin~, The Upjohn Company), and indomethacin (Indocin~,
Merck, Sharp & Dohme).
In contrast, the studies upon which the present
invention is based show that production of the various neutral
proteinases with the ability to degrade cartilage occurs even
if prostaglandin synthesis is completely blocked.
Therapeutic intervention in arthritis is hindered by
the inability to target drugs, such as the NSAIDs, to specific
areas within a mammalian host, such as, for example a joint.
Traditional routes of drug delivery, such as for example,
oral, intravenous or intramuscular a~r;n;stration, depend upon
vascular perfusion of the synovium to carry the drug to the
joint. This is inefficient because transynovial transfer of
small molecules from the synovial capillaries to the joint
space occurs generally by passive diffusion. This diffusion
is less efficient with increased size of the target molecule.
Thus, the access of large drug molecules, for example,
proteins, to the joint space is substantially restricted.
Intra-articular injection of drugs circumvents those limita-
tions; however, the half-life of drugs administered intra-
articularly is generally short. Another disadvantage of
intra-articular injection of drugs is that frequent repeated
injections are necessary to obtain acceptable drug levels at
the joint spaces for treating a chronic condition such as, for
example, arthritis. Because therapeutic agents heretofore
could not be selectively targeted to joints, it was necessary
to expose the mammalian host to systemically high
concentrations of drugs in order to achieve a sustained,
WO94t20517 21 S 7 7 8 2 PCT~S94/0~14
intra-articular therapeutic dose. Exposure of non-target
organs in this manner exacerbated the tendency of anti-
arthritis drugs to produce serious side effects, such as for
example, gastrointestinal upset and changes in the hemato-
logical, cardiovascular, hepatic and renal systems of the
mammalian host.
It has been shown that genetic material can be
introduced into mammalian cells by chemical or biologic means.
Moreover, the introduced genetic material can be expressed so
that high levels of a specific protein can be synthesized by
the host cell. Cells retaining the introduced genetic
material may include an antibiotic resistance gene thus
providing a selectable marker for preferential growth of the
transduced cell in the presence of the corresponding
antibiotic. Chemical compounds for inhibiting the production
of interleukin-1 are also known.
U.S. Patent No. 4,778,806 discloses a method of
inhibiting the production of interleukin-l by monocytes and/or
macrophages in a human by administering through the parenteral
route a 2-2'-[1,3-propan-2-onediyl-bis (thio)] bis-l H-
imidazole or a pharmaceutically acceptable salt thereof. This
patent discloses a chemical compound for inhibiting the
production of interleukin-l. By contrast, in one embodiment
of the present invention, gene therapy is employed that is
capable of binding to and neutralizing interleukin-1.
U.S. Patent No. 4,780,470 discloses a method of
inhibiting the production of interleukin-1 by monocytes in a
human by administering a 4,5-diaryl-2 (substituted) imidazole.
This patent also discloses a chemical compound for inhibiting
the production of interleukin-1.
U.S. Patent No. 4,794,114 discloses a method of
inhibiting the 5-lipoxygenase pathway in a human by
administering a diaryl-substituted imidazole fused to a
thiazole, pyrrolidine or piperidine rirg or a pharmaceutically
WOg4/20517 PCT~S94/0~14
21~7782
acceptable salt thereof. This patent also discloses a
chemical compound for inhibiting the production of
interleukin-l.
U.S. Pater.t No. 4,870,101 discloses a method for
inhibiting the release of interleukin-1 and for alleviating
interleukin-l mediated condit~ns by administering an
effective amount of a pharmaceutically acceptable anti-oxidant
compound such as disulfiram, tetrakis t3-(2,6-di-tert-butyl-4-
hydroxyphenyl) propionyloxy methyl] methane or 2,4-di-
isobutyl-~-(N,N-dimethylamino methyl)-phenol. This patent
discloses a chemical compound for inhibiting the release of
interleukin-1.
U.S. Patent No. 4,816,436 discloses a process for
the use of interleukin-1 as an anti-arthritic agent. This
patent states that interleukin-1, in association with a
pharmaceutical carrier, may be administered by intra-articular
injection for the treatment of arthritis or inflammation. In
contrast, the present invention discloses a method of using
and preparing a gene that is capable of binding to and
neutralizing interleukin-1 as a method of resisting arthritis.
U.S. Patent No. 4,935,343 discloses an immunoassay
method for the detection of interleukin-1 beta that employs a
monoclonal antibody that binds to interleukin-1 beta but does
not bind to interleukin-1 beta. This patent discloses that
the monoclonal antibody binds to interleukin-1 beta and blocks
the binding of interleukin-1 beta to nterleukin-1 receptors,
and thus blocking the biological activity of interleukin-
1 beta. The monoclonal antibody disclosed in this patent may
be obtained by production of an immunogen through genetic
engineering using recombinant DNA technology. The immunogen
is injected into a mouse and thereafter spleen cells of the
mouse are immortalized by fusing the spleen cells with myeloma
cells. The resulting cells include the hybrid continuous cell
lines (hybridomas) that may be later screened for monoclonal
WO94/20517 PCT~S94/0~14
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2157782
antibodies. This patent states that the monoclonal antibodies
of the invention may be used therapeutically, such as for
example, in the immunization of a patient, or the monoclonal
antibodies may be bound to a toxin to form an immunotoxin or
to a radioactive material or drug tc form a radio
pharmaceutical or pharmaceutical.
U.S. Patent No. 4,766,069 discloses a recombinant
DNA cloning vehicle having a DNA sequence comprising the human
interleukin-l gene DNA sequence. This patent provides a
process for preparing human interleukin-l beta, and recovering
the human interleukin-l beta. This patent discloses use of
interleukin-l as an immunological reagent in humans because of
its ability to stimulate T-cells and B-cells and increase
immunoglobulin synthesis.
U.S. Patent No. 4,396,601 discloses a method for
providing mammalian hosts with additional genetic capability.
This patent provides that host cells capable of regeneration
are removed from the host and treated with genetic material
including at least one marker which allows for selective
advantage for the host cells in which the genetic material is
capable of expression and replication. This patent states
that the modified host cells are then returned to the host
under regenerative conditions. In the present invention,
genetic material may be directly introduced (a) into host
cells in vivo or (b) into synoviocytes in vitro for subsequent
transplantation back into the patient's joints.
U.S. Patent No. 4,968,607 discloses a DNA sequence
encoding a mammalian interleukin-l receptor protein which
exhibits interleukin-l binding activity.
In spite of these prior art disclosures, there
remains a very real and substantia7 need for a method or
introducing at least one gene encoding a product into at least
one cell of a connective tissue of a mammalian host in vitro,
or alternatively ln vivo, for use in treating the mammalian
WO94/20517 PCT~S94/0~14
.
2 15 ~ 7 8
host. Further, there is a need for a process wherein a gene
encoding a truncated interleukin-1 receptor is used to resist
the deleterious pathological changes associated with
arthritis. More specifically there is a need for such a
process where a gene coding for ~he extracellular interleukin-
1 binding domain of the interleukin-1 receptor, capable of
binding to and neutralizing interleukin-1 is expressed in host
synovial cells in vivo.
SUMMA~Y OF THE INVENTION
The present invention has met the hereinbefore
described need. A method of introducing at least one gene
encoding a product into at least one cell of a connective
tissue of a mammalian host for use in treating the mammalian
host is provided for in the present invention. This method
includes employing recombinant t~c-hniques to produce a DNA
vector molecule containing the gene encoding for the product
and infecting the connective tissue cell of the mammalian host
using the DNA vector molecule containing the gene coding for
the product. The DNA vector molecule can be any DNA molecule
capable of being delivered and maintained within the target
cell or tissue such that the gene encoding the product of
interest can be stably expressed. The DNA vector molecule
preferably utilized in the present invention is either a viral
DNA vector molecule or a plasmid DNA viral molecule. This
method preferably includes introducing the gene encoding the
product into the cell of the mammalian connective tissue for
a therapeutic use.
More specifically, this method includes employing as
the gene a gene capable of encoding at least one of the
materials which is selected from the group which includes
(a) a human interleukin-l receptor antagonist protein or a
biologically active derivative or fragment thereof, (b) a
Lac Z marker gene capable of encoding a beta-galactosidase
protein or a biologically active derivative or fragment
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21~7782
thereof, (c) a soluble interleukin-l receptor protein or a
biologically active derivative or fragment thereof, (d) a
prot~;n~e inhibitor, and (e) a cytokine, and employing as the
viral vector at least one vector which is selected from the
group which includes (a) a retroviral vector including at
least one of the materials selected from the group which
includes MFG and BAG, (b) an adeno-associated virus, (c) an
adenovirus, and (d) a herpes virus, including but not limited
to herpes simplex 1 or herpes simplex 2.
A further embodiment of the present invention
includes employing as the gene a gene capable of encoding at
least one of the materials which is selected from the group
which includes (a) a human interleukin-l receptor antagonist
protein or a biologically active derivative or fragment
thereof, (b) a Lac Z marker gene capable of encoding a
beta-galactosidase protein or a biologically active derivative
or fragment thereof, (c) a soluble interleukin-l receptor
protein or a biologically active derivative or fragment
thereof, (d) a proteinase inhibitor, and (e) a cytokine, and
employing as the DNA plasmid vector any DNA plasmid vector
known to one of ordinary skill in the art capable of stable
maintenance within the targeted cell or tissue upon delivery,
regardless of the method of delivery utilized. One such
method is the direct delivery of tne DNA vector molecule,
whether it be a viral or plasmid DNA vector molecule, to the
target cell or tissue. This method also includes employing as
the gene a gene capable of encoding at least one of the
materials selected from the group which includes (a) a human
interleukin-l receptor antagonist protein or biologicallv
30 active derivative or fragment thereof, (b) a Lac Z marker gene
capable of encoding a beta-galactosidase protein or
biologically active derivative or fragment thereof, (c) a
soluble interleukin-1 receptor protein or biologically active
derivative or fragment thereof, (d) a p_oteinase inhibitor and
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215~782
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(e) a cytokine. In a specific method disclosed as an
example, and not as a limitation to the present invention, a
DNA plasmid vector containing the interleukin-1 beta coding
sequence was ligated downstream of the cytomegalovirus (CMV)
promoter. This DNA plasmid construction was encapsulated
within liposomes and injected intra-articularly into the knee
joints of recipient rabbits. Interleukin-1 beta was expressed
and significant amounts of interleukin-1 beta was recovered
from the synovial tissue. An alternative is injection of the
naked plasmid DNA into the knee joint, allowing direct
transfection of the DNA into the synovial tissue.
Another embodiment of this invention provides a
method for introducing at least one gene encoding a product
into at least one cell of a connective tissue of a mammalian
host for use in treating the mammalian host. This method
includes employing non-viral means for introducing the gene
encoding for the product into the connective tissue cell.
More specifically, this method includes employing non-viral
means which is selected from at least one of the group which
includes (a) at least one liposome, (b) Ca3 (P04) 2'
(c) electroporation, and (d) DEAE-dextran, and includes
employing as the gene a gene capable of encoding at least one
of the materials selected from the group which includes (a) a
human interleukin-1 receptor antagonist protein or
biologically active derivative or fragment thereof, (b) a
Lac Z marker gene capable of encoding a beta-galactosidase
protein or biologically active derivative or fragment thereof,
(c) a soluble interleukin-1 receptor protein or biologically
active derivative or fragment thereof, (d) a proteinase
inhibitor (e) a soluble tumor necrosis factor receptor protein
or biologically active derivative or fragment thereof, and
(f) a cytokine.
A further embodiment of this invention provides an
additional method for introducing at least one gene encoding
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a product into at least one cell of a connective tissue of a
mammalian host for use in treating the mammalian host. This
additional method includes employing the biologic means of
utilizing a virus to deliver the DNA vector molecule to the
target cell or tissue. Preferably, the virus is a
psuedovirus, the genome having been altered such that the
psuedovirus is capable only of delivery and stable maintenance
within the target cell; but not retaining an ability to
replicate within the target cell or tissue. The altered viral
genome is further manipulated by recombinant DNA techniques
such that the viral genome acts as a DNA vector molecule which
contains the heterologous gene of interest to be expressed
within the target cell or tissue. This method also includes
employing as the gene a gene capable of encoding at least one
of the materials selected from the group which includes (a) a
human interleukin-1 receptor antagonist protein or
biologically active derivative or fragment thereof, (b) a
Lac Z marker gene capable of encoding a beta-galactosidase
protein or biologically active derivative or fragmen~ thereof,
(c) a soluble interleukin-1 receptor protein or biologically
active derivative or fragment thereof, (d) a proteinase
inhibitor and (e) a cytokine.
A further embodiment of this invention includes a
method to produce an animal model for the study of connective
tissue pathology which includes introducing at least one gene
encoding a product into at least one cell of a connective
tissue of a mammalian host.
Another embodiment of this invention provides a
method of using the gene encoding an extracellular
interleukin-1 binding domain of the interleukin-1 receptor.
This gene is capable of binding to and neutralizing
interleukin-1 in vivo to substantially resist the degradation
of cartilage in a mammalian host. Unlike previous
pharmacological efforts, the method of this invention employs
WO94120~17 PCT~S94/0~14
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gene therapy in vivo to address the chronic debilitating
effects of arthritis.
A preferred method of using the gene coding for the
truncated interleukin-l receptor-of this invention involves
employing recombinant t~hn;ques to generate a cell line which
produces infectious retroviral particles containing the gene
coding for the truncated interleukin-l receptor. The producer
cell line is generated by inserting the gene coding into a
retroviral vector under the regulation of a suitable
eukaryotic promoter, transfecting the retroviral vector
containing the gene coding into the retroviral packaging cell
line for the production of a viral particle that is capable of
expressing the gene coding for the truncated interleukin-l
receptor, and infecting the synovial cells of a mammalian host
using the viral particle.
More specifically, the method of using the
hereinbefore described gene coding for the truncated
interleukin-l receptor involves introducing the viral
particles obtained from the retroviral packaging cell line
directly by intra-articular injection into a joint space of a
mammalian host that is lined with synovial cells. In a
preferred embodiment, synoviocytes recovered from the knee
joint are cultured in vitro for subsequent utilization as a
delivery system for gene therapy. It will be apparent that
Applicants are not limited to the use of the specific synovial
tissue disclosed. It would be possible to utilize other
tissue sources, such as skin cells, for in vitro culture
tec-hn;ques. The method of using the gene of this invention may
be employed both prophylactically and in the therapeutic
treatment of arthritis. It will also be apparent that
Applicants are not limited to prophylactic or therapeutic
applications in treating only the knee joint. It would be
possible to utilize the present invention either
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21~7782
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prophylactically or therapeutically to treat arthritis in any
susceptible joint.
In another embodiment of this invention, a method of
using the hereinbefore described gene coding for the truncated
interleukin-l receptor involves infecting synovial cells in
culture with the viral particles and subsequently
transplanting the infected synovial cells back into the joint.
This method of using the gene of this invention may also be
employed prophylactically and in the therapeutic treatment of
arthritis in any area susceptible to the disorder.
In another embodiment of this invention, a method of
using the gene coding for an extracellular interleukin-l
binding domain of the interleukin-l receptor that is capable
of binding to and neutralizing interleukin-l includes
employing recombinant techniques to produce a retrovirus
vector carrying two genes. The first gene encodes the
extracellular interleukin-l binding domain of the interleukin
receptor, and the second gene encodes for selectable
antibiotic resistance. This method of use involves
transfecting the retrovirus vector into a retrovirus packaging
cell line to obtain a cell line producing infectious
retroviral particles carrying the gene.
Another embodiment of this invention provides a
method of preparing a gene encoding an extracellular
interleukin-l binding domain of the interleukin-l receptor
including synthesizing the gene by a polymerase chain
reaction, introducing the amplified interleukin-l receptor
coding sequence into a retroviral vector, transfecting the
retroviral vector into a retrovirus packaging cell line and
collecting viral particles from the retrovirus packaging cell
line.
In another embodiment of this invention, a compound
for parenteral administration to a patient in a
therapeutically effective amount is provided for that contains
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2157~8~ --
a gene encoding an extracellular interleukin-l binding domain
of the interleukin-1 receptor and a suitable pharmaceutical
carrier.
Another emkodiment of this invention provides for a
compound for parenteral~a~; n; ~tration to a patient in a
prophylactically effective amount that includes a gene
encoding an extracellular interleukin-1 binding domain of the
interleukin-l receptor and a suitable pharmaceutical carrier.
An additional embodiment of the invention involves
transfection of hematopoietic progenitor cells or mature
lymphoid or myeloid cells with a DNA vector molecule
containing any of the gene or genes disclosed throughout the
specification. The transfected cells are recovered and
injected into the bone marrow of the patient using techni~ues
known and available to one of ordinary skill in the art. It
will be possible, within the scope of this method, to use
cells derived from donor bone marrow instead of cells derived
from recipient bone marrow so as to modify rejection.
In another embodiment of the invention, synoviocytes
are transfected in vivo subsequent to direct intra-articular
injection of a DNA molecule containing the gene of interest
into the joint. Transfection of the recipient synovial cells
bypasses the requirement of removal, culturing, in vitro
transfection, selection and transplanting the DNA vector
containing - synoviocytes (as disclosed in the Example
section) to promote stable expression of the heterologous gene
of interest. Methods of injecting the DNA molecule into the
joint includes, but is not limited to, encapsulation of the
DNA molecule into cationic liposomes or the direct injection
of the DNA molecule itself into the joint. The DNA molecule,
regardless of the form of presentation to the knee joint, is
preferably presented as a DNA vector molecule, either as viral
DNA vector molecule, or preferably, a DNA plasmid vector
molecule. Expression of the heterologous gene of interest is
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ensured by inserting a promoter fragment act~ve in eukaryotic
cells directly upstream of the coding region of the
heterologous gene. = One of ordinary skill in the art may
utilize known strategies and t~n;ques of vector construction
to ensure appropriate levels of expression subsequent to entry
of the DNA molecule into the synovial tissue.
It is an object of the present invention to provide
a method of introducing at least one gene encoding a product
into at least one cell of a connective tissue of a mammalian
host for use in treating the mammalian host.
It is an object of the invention to provide a method
of i~troducing a gene encoding a product into at least one
cell of a connective tissue of a mammalian host for a
therapeutic use.
It is an object of the present invention to provide
a method of introducing into the synovial lining cells of a
mammalian arthritic joint at least one gene which codes for
proteins having therapeutic properties.
It is an object of the present invention to provide
an animal model for the study of connective tissue pathology.
It is an object of the present invention to provide
a method of using in vivo a gene coding for the extracellular
interleukin-1 binding domain of the interleukin-1 receptor
that is capable of binding to and neutralizing subs'antially
all isoforms of interleukin-1, including interleukin-1 alpha
and interleukin-~ beta.
It is an object of the present invention to provide
a method of using a gene in vivo in a mammalian host that is
capable of binding to and neutralizing substantially all
isoforms of interleukin-1 and thus, substantially resist t~e
degradation of cartilage and protect surrounding soft tissues
of the joint space.
It is an object of the present invention to provide
a method of using ~n vivo a gene coding for the extracellular
WO94/20517 PCT~S9410~14
21~7~8Z
- 16 -
interleukin-l binding domain of the interleukin-l receptor
that is capable of binding to and neutralizing substantially
all isoforms of interleukin-l for the prevention of arthritis
in patients that demonstrate a high susceptibility for
developing the disease.
It is an object of the present invention to provide
a method of using in vivo a gene coding for an extracellular
interleukin-l binding domain of an interleukin-l receptor that
is capable of binding to and neutralizing substantially all
isoforms of interleukin-l for the treatment of patients with
arthritis.
It is an object of the present invention to provide
a method of using in vivo a gene or genes that address the
chronic debilitating pathophysiology of arthritis.
It is a further object of the present invention to
provide a compound for parenteral administration to a patient
which comprises a gene encoding an extracellular interleukin-l
binding domain of the interleukin-l receptor and a suitable
pharmaceutical carrier.
These and other objects of the invention will be
more fully understood from the following description of the
invention, the referenced drawings attached hereto and the
claims appended hereto.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure l shows the structure of the cDNA encoding
the human interleukin-l receptor antagonist protein (IRAP)
gene inserted into the NcoI and BamHI cloning sites of the
retroviral vector MFG.
Figure 2 shows the structure of the cDNA encoding
the human interleukin-l receptor antagonist protein (IRAP)
gene with a selectable neo marker inserted into the retroviral
vector MFG.
Figure 3 shows a micrograph of synovium recovered
from the knee of a rabbit approximately one month after
WO94/20517 PCT~S94/0~14
2157782 ~ I
intra-articular injection of Lac Z+ neo synoviocytes
employing the methods of this invention.
Figure 4 shows a Western blot demonstrating the
production of interleukin-1 receptor antagonist protein by
four cultures of HIG-82 cells (Georgescu 1988) infected using
the method of this invention employing the MFG-IRAP viral
vector.
Figure 5 shows data demonstrating the inhibition of
chondrocytes by the addition of medium conditioned by MFG-IRAP
infected HIG-82 cells.
Figure 6 shows the uptake and expression of the
Lac Z gene by synoviocytes using lipofection. Well 1 -
Control cells, treated with liposomes alone; Well 2 - Control
cells, treated with DNA alone; Well 3 - DNA + 150 nmole
liposomes; Well 4 - DNA + 240 nmole liposomes; Well 5 -
DNA + 300 nmole liposomes; Well 6 - DNA + 600 nmole
liposomes.
Figure 7 shows the interleukin-1 binding domain
amino acid arrangement.
Figures 8A-8C show the amino acid and nucleotide
sequence of the human and mouse interleukin-1 receptors.
Figure 9 shows gene encoding a truncated
interleukin-1 receptor inserted into a retroviral vector.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "patient" includes members
of the animal kingdom including but not limited to human
beings.
As used herein, the term "mammalian host" includes
members of the animal kingdom including but not limited to
human beings.
As used herein, the term "connective tissue"
includes but is not limited to a ligament, a cartilage, a
tendon, and a synovium of a mammalian host.
-
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- 18 -
As used herein, the term "DC-chol" means a cationic
liposome con~; n; ng cationic cholesterol derivatives. The
"DC-chol" molecule includes a tertiary amino group, a medium
length spacer arm (tuo atoms) and a carbamyol linker bond as
described in Biochem. Biophys. ~es. Commun., 179:280-285
(l99l), X. Gao and L. Huang.
As used herein, "SF-chol" is defined as a type of
cationic liposome.
As used herein, the term "biologically active" used
in relation to liposomes denotes the ability to introduce
functional DNA and/or proteins into the target cell.
As used herein, the term "biologically active" in
reference to a nucleic acid, protein, protein fragment or
derivative thereof is defined as an ability of the nucleic
acid or amino acid sequence to mimic a known biological
function elicited by the wild type form of the nucleic acid or
protein.
As used herein, the term "maintenance", when used in
the context of liposome delivery, denotes the ability of the
introduced DNA to remain present in the cell. When used in
other contexts, it means the ability of targeted DNA to remain
present in the targeted cell or tissue so as to impart a
therapeutic effect.
Connective tissues are difficult organs to target
therapeutically. Intravenous and oral routes of drug delivery
that are known in the art provide poor access to these
connective tissues and have the disadvantage of exposing the
mammalian host body systemically to the therapeutic agent.
More specifically, known intra-articular injection of joints
provides direct access to a joint. However, most of the
injected drugs have a short intra-articular half-life. The
present invention solves these probiems by introducing into
the connective tissue of a mammalian host genes encoding for
proteins that may be used to treat the mammalian host. More
WO94/20S17 PCT~S94/0~14
21~7782
-- 19 --
specifically, this invention provides a method for introducing
into the connective tissue of a mammalian host genes encoding
for proteins with anti-arthritic properties.
The present invention provides a method of
introducing at least one gene encoding a product into at least
one cell of a connective tissue of a mammalian host for use in
treating the mammalian host which comprises employing
recombinant tec-hniques to produce a viral vector which
contains the gene encoding for the product, and infecting the
connective tissue cell of the mammalian host using the viral
vector containing the gene coding or the product. This
method preferably includes introducing the gene encoding the
product into at least one cell of the connective tissue of the
mammalian host for a therapeutic use.
In one embodiment of this invention, the method as
hereinbefore described includes employing as the gene a gene
capable of encoding a human interleukin-1 receptor antagonist
protein (IRAP).
In another embodiment of this invention, the method
as hereinbefore described includes employing as the gene a
Lac Z marker gene capable of encoding a beta-galactosidase.
In another embodiment of this invention, the method
as hereinbefore described includes employing as the gene a
gene capable of encoding a soluble interleukin-1 receptor.
Another embodiment of this invention includes the
method as hereinbefore described including employing as the
gene a gene capable of encoding at least proteinase inhibitor.
More specifically, this method preferably includes employing
a tissue inhibitor of a metalloproteinases as the proteinase
inhibitor.
Another embodiment of this invention ir.cludes the
method as hereinbefore described including employing as the
gene a gene capable of encoding at least one cytokine. More
specifically, this method includes employing as the cytokine
WO 94/20517 PCT/US94/02414
21~7782
-- 20 --
at least one material selected from the group consisting of
interleukin-1 alpha, interleukin-1 beta, interleukin-2,
interleukin-3, interleukin-4, interleukin-5, interleukin-6,
interleukin-7, interleukin-8, interleukin-9, interleukin-10,
interleukin-11, interleukin-i2, tumor necrosis factor c~, and
tumor necrosis factor B.
A further embodiment of this invention includes a
method as hereinbefore described including employing as the
cytokine at least one transforming growth factor. More
specifically, this method includes employing as the trans-
forming growth factor a growth factor selected from the group
consisting of TGF-betal, TGF-beta2, TGF-beta3, and TGF-alpha.
Each transforming growth factor is commercially available from
R & D Systems, 614 McKinley Place, N.E., Minneapolis, MN
55413.
In another embodiment of this invention, the method
as hereinbefore described includes employing as the cytokine
at least one fibroblast growth factor The fibroblast growth
factors are also commercially available from R & D Systems,
614 McKinley Place, N.E., Minneapolis, MN 55413.
Another embodiment of this invention includes the
method as hereinbefore described including employing as the
viral vector a retroviral vector. More specifically, this
method includes employing as the retroviral vector at least
one material selected from the group consisting of MFG and
BAG. A preferred embodiment of this invention includes
providing the method as hereinbefore described including
employing as the gene a gene capable of encoding a human
interleukin-1 receptor antagonist protein and employing MFG as
the retroviral vector.
Another preferred embodiment of this invention
includes the method as hereinbefore described including
employing a Lac Z marker gene as the gene capable of encoding
WO94120517 PCT~S94/0~14
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- 21 -
a beta-galactosidase and employing MFG as the retroviral
vector.
Another preferred embodiment of this invention
provides the method as hereinbefore described including
employing a Lac Z neo marker gene as the gene capable of
encoding a beta-galactosidase and employing BAG as the
retroviral vector.
In a most preferred embo~;rent of this invention,
the method as hereinbefore described includes employing a
retroviral vector selected from the group consisting of MFG
and BAG and includes employing as the gene a gene capable of
encoding a soluble interleukin-l receptor.
In another embodiment of this invention, a method as
hereinbefore described is provided including employing as the
gene a gene capable of encoding at least one proteinase
inhibitor and including employing as the retroviral vector at
least one material selected from the group consisting of MFG
and BAG.
In another embodiment of this invention, a method as
hereinbefore described is provided which includes employing as
the retroviral vector at least one material selected from the
group consisting of MFG and BAG and including employing as the
gene a gene capable of encoding at least one cytokine as
hereinbefore described.
In another embodiment of this invention, a method is
provided for introducing at least one gene encoding a product
into at least one cell of a connective tissue of a mammalian
host for use in treating the mammalian host which compr ses
employing recombinant techniques to produce a viral vector
which contains the gene encoding for the product and infecting
the connective tissue cell of the mammalian host using the
viral vector containing the gene coding for the product,
wherein the viral vector is at least one vector selected from
the group consisting of an adeno--~ssociated virus, an
WO94/20517 PCT~S94/0~14
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adenovirus, and a herpes virus, such as herpes simplex type-1
or herpes simplex type-2.
This method includes employing as the gene a gene capable of
encoding at least one material selected from the group which
includes (a) a human interleukin-1 receptor antagonist
protein, (b) a soluble interleukin-1 receptor, (c) a Lac Z
marker gene capable of encoding a beta-galactosidase, (d) at
least one proteinase inhibitor and (e) at least one cytokine.
More specifically, this method includes employing a tissue
inhibitor of metalloproteinases as the proteinase inhibitor
and includes employing as the cytokine at least one of the
materials selected from the group which includes (a) at least
one transforming growth factor selected from the group
consisting of TGF-beta" TGF-beta2, TGF-beta3, and TGF-alpha,
(b) at least one fibroblast growth factor,
(c) interleukin-1 alpha, (d) interleukin-l beta,
(e) interleukin-2, (f) interleukin-3, (g) interleukin-4,
(h) interleukin-5, (i) interleukin-6, (j) interleukin-7,
(k) interleukin-8, (l) interleukin-9, (m) interleukin-10,
(n) interleukin-11, and (o) interleukin-12 (p) tumor necrosis
factor a, and (q) tumor necrosis factor B.
Another embodiment of this invention includes the
method as hereinbefore described including introducing the
gene into a connective tissue which tissue is selected from
the group consisting of a ligament, a cartilage, a tendon, and
a synovium. It is preferable that this method includes
employing a cruciate ligament as the ligament. Most prefer-
able this method includes employing as the cruciate ligament
a ligament selected from the group consisting of an anterior
cruciate ligament and a posterior cruciate ligament.
Another embodiment of this invention includes the
method as hereinbefore described including employing as the
gene a gene having DNA that is capable of maintenance and
expression.
WO94/20517 PCT~S94/0~14
~ 21S7782
A further embodiment of this inventior. includes the
method as hereinbefore described including introducing the
gene into the cell in vitro. This method includes
subsequently transplanting the infected cell into the
mammalian host. This method also includes after effecting the
infecting of the connective tissue cell but before the
transplanting of the infected cell into the mammalian host,
storing the infected connective tissue cell. It will be
appreciated by those skilled in the art that the infected
connective tissue cell may be stored frozen in 10 percent DMSO
in liquid nitrogen. This method inc udes employing a method
to substantially prevent the development of arthritis in a
mammalian host having a high susceptibility of developing
arthritis.
The method of this invention includes employing the
method on an arthritic ~rAlian host for a therapeutic use.
This method includes employing a method to repair and
regenerate the connective tissue whicn tissue is selected from
the group consisting of a ligament, a cartilage, a tendon, and
a synovium. This method includes employing the method on a
m~Alian host that is a human being.
Another embodiment of this invention includes a
method of introducing at least one gene encoding a product
into at least one cell of a cor.nective tissue of a mammalian
host for use in treating the mammalian host as hereinbefore
described including effecting in vivo the infection of the
cell by introducing the viral vector containing the gene
coding for the product directly into the mammalian host.
Pre~erably, this method includes effecting the direct
introduction into the mammalian host by intra-articular
injection. This method includes employing the method to
substantially prevent a developmen' of arthritis in
mammalian host having a high susceptibility of developing
arthritis. This method also includes employing the method on
WO94/20517 PCT~S94/0~14
2l~7~82
an arthritic mammalian host for therapeutic use. Further this
method as includes employing the method to repair and
regenerate the connective tissue a$ hereinbefore defined.
In yet another embo~ rnt of this invention, a
method of introducing at least one gene encoding a product
into at least one cell of a connective tissue of a mammalian
host for use in treating the mammalian host includes employing
non-viral means for introducing the gene encoding for the
product into the connective tissue cell. This method includes
employing non-viral means selected from the group consisting
of at least one liposome, Ca3(PO4)2, electroporation, and
DEAE-dextran. This method includes employing as the liposome
a material selected from the group consisting of DC-chol and
SF-chol.
It will be understood that the method of this
invention of introducing at least one gene encoding a product
into at least one cell of a connective tissue of a mammalian
host for use in treating the mammalian host that includes
employing non-viral means for introducing the gene encoding
for the product into the connective tissue cell is a non-
infectious delivery system. An advantage of the use of a non-
infectious delivery system is the elimination of insertional
mutagenesis and virally induced disease.
It will be appreciated by those skilled in the art,
that the viral vectors employing a liposome are not limited by
cell division as is required for the retroviruses to effect
infection and integration of connective tissue cells. This
method employing non viral means as hereinbefore described
includes employing as the gene a gene capable of encoding at
least one of the following materials selected from the group
which includes (a) a human interleukin-1 receptor antagonist
protein, (b) a Lac Z marker gene capable of encoding a
beta-galactosidase, (c) a soluble interleukin-l receptor,
(d) at least one proteinase inhibitor, (e) at least one
.
WO94/20517 PCT~S94/0~14
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- 25 -
transforming growth factor, and (f) at least one cytokine.
More specifically, this method includes employing as the
cytokine a cytokine selected from the group which includes
interleukin-l alpha, interleukin-l beta, interleukin-2,
interleukin-3, interleukin-4, interleukin-5, interleukin-6,
interleukin-7, interleukin-8, interleukin-9, interleukin-lO,
interleukin-ll, interleukin-12, tumor necrosis factor ~, tumor
necrosis factor B, at least one fibroblast growth factor, and
at least one transforming growth factor. Preferably, this
method includes employing as the transforming growth factor a
growth factor selected from the group consisting of TGF-betal,
TGF-betal, TGF-beta3, and TGF-alpha.
Another preferred embodiment of this invention
includes providing the method employing non-viral means as
hereinbefore described which includes employing a tissue
inhibitor of metalloproteinases as the proteinase inhibitor.
This method employing non-viral means for introducing the gene
encoding for the product into the connective tissue cell as
hereinbefore described includes introducing the gene into the
connective tissue which tissue is selected from the group
consisting of a ligament, a cartilage, a tendon, and a
synovium. Preferably, this method includes employing a
cruciate ligament as the ligament. The cruciate ligament is
selected from the group consisting of an anterior cruciate
ligament and an posterior cruciate ligament.
Another embodiment of this invention provides the
method of introducing at least one gene encoding a product
into at least one cell of a connective tissue of a mamma;ian
host for use in treating the mammalian host which includes
employing non-viral means as hereinbefore described and
includes employing as the gene a gene having DNA that is
capable of maintenance and expression.
In yet a further embodiment of this invention, the
method of introducing at least one gene encoding a product
WO94/20517 PCT~S94/0~14
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- 26 -
into at least one cell of a connective tissue of a mammalian
host for use in treating the mammalian host is provided that
includes employing non-viral means for introducing the gene
encoding for the product into the connective tissue cell
n vitro and includes subsequently transplanting the cell
having the gene into the mammalian host. Another embodiment
of this invention provides a method including after
introducing the gene encoding for the product in the
connective tissue cell and before the transplanting of the
connective tissue cell having the gene into the mammalian
host, storing the connective tissue cell having the gene.
This method includes storing connective tissue cell frozen in
lO percent DMS0 in liquid nitrogen. This method includes
employing a method to substantially prevent the development of
arthritis in a mammalian host having a high susceptibility of
developing arthritis. Further, this method includes employing
the method on an arthritic mammalian host for a therapeutic
use. This method includes employing the method to repair and
regenerate the connective tissue which tissue is selected from
the group consisting of a ligament, a cartilage, a tendon, and
a synovium.
A further embodiment of this invention provides a
method of introducing at least one gene encoding a product
into at least one cell of a connective tissue of a mammalian
host for use in treating the mammalian host which includes
employing non-viral means in vivo for directly introducing the
gene encoding for the product into the connective tissue cell
of the mammalian host. The non-viral means is selected from
the group consisting of at least one liposome ~ ca3 (P04) 2 and
DEAE-dextran. Preferably, this method includes effecting the
in vivo introduction into the mammalian host by
intra-articular injection. This method includes employing the
method to substantiall~ prevent the development of arthritis
in a mammalian host having a high susceptibility of developing
W094120517 PCT~S94/0~14
21577~o~
- 27 -
arthritis. Further, this method includes employlng the method
on an arthritic mammalian host for a therapeutic use. This
method also includes employing the method to repair and
regenerate the connective tissue which tissue is selected from
the group consisting of a ligament, a cartilage, a tendon, and
a synovium.
Another embodiment of the present invention is a
method to produce an animal model for the study of connective
tissue pathology. As will be understood by those skilled in
the art, over-expression of interleukin-l in the joint of a
mammalian host is generally responsible for the induction of
an arthritic condition. This invention provides a method for
producing an animal model using the hereinbefore described
gene transfer technology of this invention. Preferably, the
method of this invention provides a method for producing an
animal model using the hereinbefore described gene transfer
technology of this invention to effect an animal model for
arthritis. For example, constitutive expression of
interleukin-l in the joint of a rabbit following the method of
gene transfer provided for by this invention leads to the
onset of an arthritic condition. It will be appreciated by
those skilled in the art that this rabbit model is suitable
for use for the testing of therapeutic agents. This method
includes introducing at least one gene encoding a product into
at least one cell of a connective tissue of a ~r~lian host
comprising (a) employing reccmbinant techniques to produce a
viral vector which contains the gene encoding for the product
and (b) infecting the connective tissue cell of the ~rr~lian
host using the viral vector containing the gene coding for the
product for effecting the animal model. This method includes
employing as the gene a material selected from the group
consisting of a cytokine and a proteinase. This method
includes employing as the cytokine a material selected from
the group consisting of interleukin-l alpha,
WO94120517 PCT~S94/0~14
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- 28 -
interleukin-l be_a, and tumor necrosis factor-~ (TNF-~). This
method includes employing as the proteinase a matrix
metalloproteinase. The matrix metalloprGteinase is an enzyme
selected from the group consisting of a collagenase, a
gelatinase and a stromelysin. It will be apparent that use of
the term "a collogenase, a gelatinase and a stromolysin" is
meant to include the plural, and not be limited to the
singular. It is well known in the art that numerous
collagenases, gelatinases and stromolysins could be employed
as a matrix metalloproteinase in the present invention. A
further embodiment of this invention provides a method to
produce an animal model for the study of connective tissue
pathology which includes employing non-viral means for
introducing at least one gene encoding a product into at least
lS one cell of a connective tissue of a mammalian host for
effecting the animal model. The non-viral means is selected
from the group consisting of at least one liposome, Ca3(PO4)2,
electroporation, and DEAE-dextran. This method includes
employing as the gene a material selected from the group
consisting of a cytokine and a proteinase. This method
includes employing as the cytokine a material selected from
the group consisting of interleukin-l alpha,
interleukin-l beta, and TNF-~. This method also includes
employing as the proteinase a matrix metalloproteinase. The
matrix metalloproteinase includes an enzyme selected from at
least one of the group consisting of a collagenase, a
gelatinase, and a stromelysin.
A further embodiment of the present invention
includes employing as the gene a gene capable of encoding at
least one of the materials which is selected from the group
which includes (a~ a human interleukin-l receptor antagonist
protein or a biologically active derivative or fragment
thereof, (b) a Lac Z marker gene capable of encoding a
beta-galactosidase protein or a biologically active derivative
WO94/20517 PCT~S94/0~14
~1~7782
- 29 -
or fragment thereof, (c) a soluble interleukin-l receptor
protein or a biologically active derivative or fragment
thereof, (d) a proteinase inhibitor, (e) a soluble tumor
necrosis factor receptor protein or a biologically active
derivative or fragment thereof and (f) a cytokine, and
employing as the DNA vector any DNA vector, preferably a
plasmid or viral vector, known to one of ordinary skill in the
art capable of stable maintenance within the targeted cell or
tissue upon delivery, regardless of the method of delivery
utilized. In one embodiment of the invention, synoviocytes
are transfected n vivo subsequent to direct intra-articular
injection of a DNA molecule containing the gene of interest
into the joint. Transfection of the recipient synovial cells
bypasses the requirement of removai, culturing, in vitro
transfection, selection and transplanting the DNA vector
containing - synoviocytes (as disclosed in the Example
section) to promote stable expression of the heterologous gene
of interest. Methods of injecting the DNA molecule into the
joint includes, but is not limited to, encapsulation of the
DNA molecule into cationic liposomes or the direct injection
of the DNA molecule itself into the joint. Expression of the
heterologous gene of interest subsequent to n vivo
transfection of the synovial tissue is ensured by inserting a
promoter fragment active in eukaryotic cells directly upstream
of the coding region of the heterologous gene. One of
ordinary skill in the art may utilize known strategies and
t~chn;ques of vector construction to ensure appropriate levels
of expression subsequent to entry of the DNA molecule into the
synovial tissue. As an example, and not a limitation, of the
present invention, a DNA plasmid vector containing the
interleukin-l beta coding sequence ligated downstream of the
- CMV promoter was encapsulated within liposomes and injected
into the knee joints of recipient rabbits. Interleukin-l beta
was expressed in synovial tissue, as significant amounts of
WO94/20517 PCT~S94/0~14 ~
21S~ ~ 82
- 30 -
interleukin-l beta was recovered from the synovial tissue
within the region of intra-articular injection.
A further embodiment~ of this invention provides an
additional method for introdùcing at least one gene encoding
a product into at least one cell of a connective tissue of a
mammalian host for use in treating the mammalian host. This
additional method includes employing the biologic means of
utilizing a virus to deliver the DNA vector molecule to the
target cell or tissue. Preferably, the virus is a
psuedovirus, the genome having been altered such that the
psuedovirus is capable only of delivery and stable maintenance
within the target cell; but not retaining an ability to
replicate within the target cell or tissue. The altered viral
genome is further manipulated by recombinant DNA techniques
such that the viral genome acts as a DNA vector molecule which
contains the heterologous gene of interest to be expressed
within the target cell or tissue. This method also includes
employing as the gene a gene capable of encoding at least one
of the materials selected from the group which includes (a) a
human interleukin-1 receptor antagonist protein or
biologically active derivative or fragment thereof, (b) a
Lac Z marker gene capable of encoding a beta-galactosidase
protein or biologically active derivative or fragment thereof,
(c) a soluble interleukin-1 receptor protein or biologically
active derivative or fragment thereof, (d) a proteinase
inhibitor and (e) a soluble tumor necrosis factor receptor
protein or a biologically active derivative or fragment
thereof and ~f) a cytokine.
The following examples are offered by way of
illustration of the present inventicn, and not by way of
limitation.
WO94/20517 PCT~S94/0~14
~IS~:~8~
EXAMPLE I
Packaginq of AAV
The only cis-acting sequences required for
replication and packaging of recombinant adeno-associated
virus (AAV) vector are the AAV terminal repeats. Up to 4 kb
of DNA can be inserted between the terminal repeats without
effecting viral replication or packaging. The virus rep
proteins and viral capsid proteins are required in trans for
virus replication as is an adeno-associated virus helper. To
package a recombinant AAV vector, the plasmid containing the
terminal repeats and the therapeutic gene is co-transfected
into cells with a plasmid that expresses the rep and capsid
proteins. The transfected cells are then infected with
adeno-associated virus and virus isolated from the cells about
48-72 hours post-transfection. The supernatants are heated to
about 56 Centigrade to inactivate the adeno-associated virus,
leaving a pure virus stock of recombinant AAV.
EXAMPLE II
Electro~oration
The connective tissue cells to be electroporated are
placed into Hepes buffer saline (HBS) at a concentration of
about 107 cells per ml. The DNA to be electroporated is added
at a concentration of about 5-20 ug/ml of HBS. The mixture is
placed into a cuvette and inse~ted into the cuvette holder
that accompanies the Bio-RAD electroporation device (1414
Harbour Way South, Richmond, CA 94804). A range between about
250 and 300 volts at a capacitance of about 960 ufarads is
required for introduction of DNA into most eukaryotic cell
types. Once the DNA and the cells are inserted into the
Bio-RAD holder, a button is pushed and the set voltage is
delivered to the cell-DNA solution. The cells are removed
~ from the cuvette and replated on plastic dishes.
WOg4/20517 ~ PCT~S9~/0~14
Z1577~2
- 32 -
EXAMPLE III
The cDNA encoding the human interleukin-1 receptor
antagonist (IRAP) was inserted into the NcoI and BamHI cloning
sites of the retroviral véctor MFG as shown in Figure 1.
Specifically, a Pstl to Ba~HI fragmen' from the IRAP cDNA was
linked to a synthetic oligonucleotide adapter from the NcoI
site (representing the start site of translation for IRAP) to
the Pstl site (approximately 12 base pairs downstream from the
NcoI site) to the MFG backbone digested at NcoI and BamHI in
a three part ligation reaction. This three part ligation
involving a synthetic oligo and two DNA fragments is well
known by those skilled in the art of cloning. LTR means long
terminal repeats, 5'SD means 5' splice donor, 3'SA means 3'
splice acceptor. The straight arrow and the crooked arrow in
Figure 1 represent unspliced and spliced messenger RNAs
respectively. IRAP is encoded by the spliced message.
Figure 2 shows the cDNA encoding the human
interleukin-1 receptor antagonist protein (IRAP) with a
selectable neo gene marker. Figure 3 shows a low power
micrograph of synovium recovered from the knee of a rabbit one
month after intra-articular injection of Lac Z+,
neo+ synoviocytes. Tissue was stained histochemically for the
presence of beta galactosidase. This micrograph
counterstained with eosin revealed an area of intensely
stained, transplanted cells demonstrating that these cells
have colonized the synovial lining of the recipient joint.
EXAMPLE IV
Animal Models
The methods of this invention of transferring genes
to the synovia of mammalian joints permit the production and
analysis of joint pathologies that were not previously
possible. This is because the only other way of delivering
potentially arthriotogenic compounds to the joint is by
intra-articular injection. Not only are such compounds
WO94120517 PCT~S94/0~14
~ 21~7782
quickly cleared from joints, but the effects of bolus
injections of these compounds do not accurately mimic
physiological conditions where they are constantly produced
over a long period of time. In contrast, the gene transfer
technologies of this invention permit selected proteins of
known or suspected involvement in the arthritic process to be
expressed intra-articularly over an extended period of time,
such as for example, at least a three month period. The
animal models of this invention therefore permits the
importance of each gene product to the arthritic process to be
evaluated individually. Candidate genes include, but are not
restricted to, those coding ~or cytokines such as
interleukin-l (IL-l) alpha, IL-l beta, and TNF-alpha, and
matrix metalloproteinases such as collagenases, gelatinases
and stromelysins.
Additionally, the gene transfer techniques of this
invention are suitable for use in the screening of potentially
therapeutic proteins. In this use, the animal models of the
invention are initiated in joints whose synovia express gene
coding for potential anti-arthritic proteins. Candidate
proteins include, but are not restricted to, inhibitors of
proteinases such as, for example, the tissue inhibitor of
metalloproteinases, and cytokines such as, for example, trans-
forming growth factor-beta.
EXAMPLE V
Method For Using Synoviocytes As A Delivery System
For Gene TherapY
Rabbits are killed by intravenous injection of 4 ml
nembutol, and their knees quickly shaved. Synovia are
surgically removed from each knee under aseptic conditions !
and the cells removed from their surrounding matrix by
- sequential digestion with trypsin and collagenase (0.2% w/v in
Gey's Balanced Salt Solution) for about 30 minutes and about
2 hours, respectively. The cells recovered in this way are
WO94/20517 PCT~S94/0~14 ~
215~8~
seeded into 25 cm2 culture flasks with about 4 ml of Ham's Fl2
nutrient medium supplemented with 10% fetal bovine serum,
about 100 U/ml penicillin and about 100 ) g/ml streptomycin,
and incubated at about 37 in an atmosphere of 95% air, 5% CO2.
Following about 3-4 days incubation, the cells attain
confluence. At this stage, the culture medium is removed and
the cell sheet washed twice with approximately 5 mls of Grey's
Balanced Salt Solution to remove non-adherent cells such as
lymphocytes. The adherent cells are then treated with ~rypsin
(0.25% w/v in balanced salt solution). This treatment
detaches the fibroblastic, Type B synoviocytes, but leaves
macrophages, polymorphonuclear leukocytes and the Type A
synoviocytes attached to the culture vessel. The detached
cells are recovered, re-seeded into 25 cm2 culture vessels at
a 1:2 split ratio, medium is added and the culture returned to
the incubator. At confluence this procedure is repeated.
After the third such passage, the cells are
uniformly fibroblastic and comprise a homogeneous population
of Type B synoviocytes. At this stage, cells are infected
with the retroviral vector.
Following infection, cells are transferred to fresh
nutrient medium supplemented with about 1 mg/ml G418
(GIBCO/BRL, P.O. Box 68, Grand Island, NY 14072-0068)
and returned to the incubator. Medium is changed every three
days as neo- cells die and the neo+ cells proliferate and
attain confluency. When confluent, the cells are trypsinized
and subcultured as described above. One flask is set aside
for staining with X-gal to confirm that the neo+ cells are
also Lac Z+. When the subcultures are confluent, the medium
is recovered and tested for the presence of IRAP, soluble
IL-lR or other appropriate gene products as hereinbefore
described. Producing synoviocyte cultures are then ready for
transplantation.
~ WO94/20517 PCT~S94/0~14
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- 35 -
The day before transplantation, the cells are
recovered by trypsinizing, as hereinbefore described. These
cells are then suspended in nutrient medium, and incubated
overnight in an untreated plastic centrifuge tube. Under
these conditions, the cells do not adhere, bu. they regenerate
their cell surface proteins that were removed by trypsinizing.
The following morning, the cells are recovered by
centrifuging, washed several times by resuspension in Gey's
Balanced Salt Solution and finally resuspended at a concentra-
tion of about 106-107 cells/ml in Grey's solution.
Approximately 1 ml of this suspension is then introduced into
the knee joint of a recipient rabbit by intra-articular
injection. For this purpose a 1 ml syringe with a 25-gauge
hypodermic needle is used. Injection is carried out through
the patellar tendon. Experiments in which radioopaque dye was
injected have confirmed that this method successfully intro-
duces material into all parts of the joint.
EXAMPLE VI
The method of Example V for producing generally
uniformly fibroblastic cells of a homogeneous population of
Type B synoviocytes was followed to effect growing cultures of
lapine synovial fibroblasts. These growing cultures of lapine
synovial fibroblasts were subsequently infected with an
amphotropic retroviral vector carrying marker genes coding for
beta-galactosidase (Lac Z) and resistance to the neomycin
analogue G418 (neo+). Following infection and growth in
selective medium containing about 1 mg/ml G418, all cells
stained positively in a histochemical stain for beta-
galactosidase.
- 30 Neo selected cells carrying the Lac Z marker gene
were transplanted back into the knees of recipient rabbits to
examine the persistence and expression of these genes in vivo.
Two weeks following transplantation, islands of Lac Z+ cells
within the synoviu~. of recipient knees were observed. This
WO94/20517 PCT~S94/0~14 ~
~ 2~5~
- 36 -
confirmed the akility of the method of this invention to
introduce marker genes into rabbit synovia and to express them
n situ.
EXAMPLE VII
Neo-selected, Lac Z+ synoviocytes were recovered
from cell culture, suspended in Gey's Balanced Salt Solution
and injected intra-articularly into the knee joints of
recipient rabbits (about lOs _ 107 cells per knee). Contra-
lateral control knees received only a carrier solution. At
intervals up to 3 months following transplant, the rabbits
were killed and their synovia and surrounding capsule
recovered. Each sample may be analyzed in three ways. A
third of the synovium was stained histochemically en masse for
the presence of beta-galactosidase. A second portion may be
lS used for immunocytochemistry using antibodies specific for
bacterial beta-galactosidase. The final portion may be
digested with trypsin and collagenase, and the cells thus
recovered cultured in the presence of G418.
Staining of the bulk synovial tissue revealed
extensive areas of Lac Z+ cells, visible to the naked eye.
Control synovia remained colorless. Histochemical examination
of synovia revealed the presence of islands of cells staining
intensely positive for beta-galactosidase. These cells were
present on the superficial layer of the synovial lining, and
were absent from control synovia. From such tissue it was
possible to grow Lac Z+, neo+ cells. Cells recovered from
control tissue were Lac Z~ and died when G418 was added to the
culture. This indicates that the transplanted, transduced
synovial fibroblasts have successfully recolonized the synovia
of recipient joints, and continue to express the two marker
genes, Lac Z and neo. Maintaining intra-articular Lac Z and
neo expression in transplanted synoviocytes has been effected
for 3 months using primary cells and one month using the
HIG-82 cell line.
~ WO94/20517 PCT~S94/0~14
21~7782
- 37 -
EXAMPLE VIII
Based upon the methods of the hereinbefore presented
examples, and employing stAn~rd recombinant techniques well
known by those skilled in the art, the human IRAP gene was
incorporated into an MFG vector as shown in Figure 1.
Following the infection of synoviocyte cultures of rabbit
origin with this viral vector, IRAP was secreted into the
culture medium.
Western blotting, well known by those skilled in the
art, was carried out using an IRAP-specific rabbit polyclonai
antibody that does not recognize human or rabbit IL-1 alpha or
IL-1 beta, or rabbit IRAP. Figure 4 shows a Western blot
which sets forth the production of IRAP by four cultures of
HIG-82 cells infected with MFG-IRAP. Three forms of the IRAP
are present: a non-glycosylated form which runs with
recombinant standards, and two larger glycosylated forms. The
results of the Western blotting shown in Figure 4 demonstrated
that IRAP was produced by HIG-82 synoviocyte cell line
(Georgescu, 1988) following infection with the MFG-IRAP vector
of this invention. The Western blotting of Figure 4 shows the
IRAP concentration of the conditioned medium is as high as
50 ng/ml. This is approximately equal to 500 ng IRAP/106
cells/day. Lane 1 and Lane 2 of Figure 4 show that the
recipient synovia tissue secrete substantial amounts of HIG-
IRAP at 3 days (Lane 2) and 6 days (Lane 1). Lane 3 shows
human recombinant IRAP. Lane 6 indicates that rabbit synovial
cells produce a larger glycosylated version of this molecule
after infection with MFG-IRAP. Lane 7 indicates that native
rabbit synovial celis do not produce this glycosylated form.
Figure 5 shows that medium conditioned by IRAP+
synoviocytes blocks the induction of neutral metallopro-
teinases in articular chondrocytes exposed to recombinant
human IL-l beta. Chondrocytes normally secrete 1 U/106 cells,
or less, gelatinase into their culture media. Figure 5 shows
WO94/20517 PCT~S94/0~14 ~
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- 38 -
that when to about 5 U/ml or lO U/ml IL-1 are added,
gelatinase production increases to over 4 U and 6U/108 cells,
respectively. Addition of medium conditioned by MFG-IRAP-
infected HIG-82 cells employed by the method of this invention
suppressed gelatinase production by IL-1 treated chondrocytes.
With 5 U/ml IL-l (Figure 5, right panel) inhibition was 100%
for one culture and 41% for the other. With 10 U/ml IL-l,
inhibition was reduced to 38% and 18% (Figure 5, left panel)
as is expected of a compeiitive inhibitor. These data
demonstrate that the IRAP produced by HIG-82 cells infected
with MFG-IRAP is biologically active.
EXAMPLE IX
This example demonstrates the uptake and expression
of Lac Z gene by synoviocytes using infection by a liposome
(lipofection). A six well plate containing synoviocyte
cultures were transduced with the Lac Z gene by lipofection.
The content of each well is as follows:
Well 1 Control cells, treated with liposomes alone
Well 2 Control cells, treated with DNA alone
Well 3 DNA + 150 nmole liposomes
Well 4 DNA + 240 nmole liposomes
Well 5 DNA + 300 nmole liposomes
Well 6 DNA + 600 nmole liposomes
Wells 3-6 containing sub-confluent cultures of synovial
fibroblasts were infected with 6 ug of DNA complexed with
150-600 nmoles/well of "DC-chol" liposome or in the
alternative, with "SF-chol". Three days later, cells were
stained histochemically for expression of beta-galactosidase
(Figure 6).
Table 1 shows the results Gf using the liposomes
"DC-chol" and "SF-chol" in converting synoviocyte cultures to
the Lac Z+ phenotype without selection. Table 1 sets forth
that the "DC-chol" liposome in a concentration of about 300
nmole/well converted generally 30% of the synovial cells in
WO94/20517 PCT~S94/0~14
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- 39 -
synoviocyte cultures to the Lac Z+ phenotype without
selection. Reduced expression was shown in Well 6 for
"DC-chol" due to the toxic effect of the high liposome
concentration.
TABLE 1
% Lac Z+ Cells
Liposome,
nmole/wellDC-chol SF-chol
150 10 0.5
240 22 1.0
300 30 2.8
600 NA 3.5
In another embodiment of this invention, a gene and
method of using this gene provides for the neutralization of
interleukin-l. Interleukin-l is a key mediator of cartilage
destruction in arthritis. Interleukin-l also causes
inflammation and is a very powerful inducer of bone
resorption. Many of these effects result from the ability of
interleukin-l to increase enormously the cellular synthesis of
prostaglandin E2, the neutral proteinases -- collagenase,
gelatinase, and stromelysin, and plasminogen activator. Th~
catabolic effects of interleukin-l upon cartilage are
exacerbated by its ability to suppress the synthesis of the
cartilaginous matrix by chondrocytes. Interleukin-l is
present at high concentrations in synovial fluids aspirated
from arthritic joints and it has been demonstrated that
intra-articular injection of recombinant interleukin-l in
animals causes cartilage breakdown and inflammation.
Interleukin-1 exists as several species, such as
unglycosylated polypeptide of 17,000 Daltons. Two species
have previously been cloned, interleukin-l alpha and
WO94/20517 PCT~S94/0~14 ~
21S7~82
- 40 -
interleukin-l beta. The alpha form has a pI of approximately
5, and the beta form has a pI aro`und 7. Despite the existence
of these isoforms, interleukin-l alpha and interleukin-1 beta
have substantially identical biological properties and share
common cell surface receptors. The type I interleukin-1
receptor is a 80kDa (kilodalton) glycoprotein and contains an
extracellular, interleukin-l binding portion of 319 amino
acids which are arranged in three immunoglobulin-like domains
held together by disulfide bridges as shown in Figure 7. A 21
amino acid trans-membrane domain joins the extracellular
portion to the 217 amino acid cytoplasmic domain. Figures
8A-8C show the amino acid and nucleotide sequence of the human
and mouse interleukin-1 receptors. In Figure 8B, the 21 amino
acid trans-membrane region of the interleukin-l receptor is
marked by the thicker solid line. In Figures 8A and 8B, the
position of the 5' and 3' oligonucleotides for PCR are marked
by thinner short lines, respectively. The lysine amino acid
just 5' to the trans-membrane domain to be mutated to a stop
codon is marked by a solid circle in Figure 8B.
Synovium is by far the major, and perhaps the only,
intra-articular source of interleukin-1 in the arthritic
joint. Snyovia recovered from arthritic joints secrete high
levels of interleukin-l. Both the resident synoviocytes and
infiltrating blood mononuclear cells within the synovial
lining produce interleukin-1.
The present invention provides a method of using in
vivo a gene coding for a truncated form of the interleukin-1
receptor which retains its ability to bind interleukin-1 wi~h
high affinity but which is released extracellularly and
therefore inactive in signal transduction. The binding of
this truncated and modified receptor to interleukin-1 inhibits
the intra-articular activity of interleukin-1.
This method of using a gene encoding the
extracellular interleukin-1 binding domain of an interleukin-1
WO94/20517 - PCT~S94/0~14
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- 41 -
receptor that is capable of binding to and neutralizing
interleukin-l includes employing a retroviral vector carrying
a truncated interleukin-1 receptor gene which encodes a
truncated and soluble active form of the receptor. The
expression of the novel interleukin-1 receptor gene is
controlled by regulatory se~l~nc~c contained within the vector
that are active in eukaryotic cells. This recombinant viral
vector is transfected into cell lines stably expressing the
viral proteins in trans required for ~roduction of infectious
virus particles carrying the recombinant vector. These viral
particles are used to deliver the recombinant interleukin-1
receptor to the recipient synovial cells by direct virus
infection in vivo.
The soluble human interleukin-1 receptor to be
inserted into the retroviral vector may be generated by a
polymerase chain reaction (PCR). An oligonucleotide
complementary to the 5' leader sequence of the human
interleukin-1 receptor (GCGGATCCCCTCCTAGAAGCT) and an
oligonucleotide complementary to a region just upstream from
the transmembrane domain of the interleukin-l receptor
(GCGGATCCCATGTGCTACTGG) are used as primers for PCR. The
primer for the region of the interleukin-1 receptor adjacent
to the trans-membrane domain contains a single base change so
that the lys codon at amino acid 319 (AAG) is changed to a
stop codon (TAG). By inserting a translation stop codon just
upstream from the transmembrane domain, a truncated form of
interleukin-1 receptor that is secreted by the cell is
generated. A BamHI recognition sequence (GGATCC) is added to
the 5' end of the PCR primers, and following amplification,
the resulting interleukin-1 receptor fragment is cloned into
a BamHI site. A cDNA library from human T-cells is used as a
source for the interleukin-1 receptor cDNA. To amplify the
appropriate region of the interleukin-1 receptor from the cDNA
library, the complementary primers are added to the DNA and 50
WO94/20517 PCT~S94/0~14 ~
~S~2
- 42 -
cycles of annealing, primer extension and denaturation are
performed using a thermocycler and standard PCR reaction
conditions well known by tho~e~ persons skilled in the art.
Following amplification of t~e interleukin-1 soluble receptor
using the PCR process, the resulting fragment is digested with
BamHI and inserted into the pLJ retroviral vector. The pLJ
retroviral vector is available from A. J. Korman and R. C.
Mulligan. See also Proc. Natl. Acad. Sci., Vol. 84, pp. 2150-
2154 (April 1987) co-authored by Alan J. Korman, J. Daniel
Frantz, Jack L. Strominger and Richard C. Mulligan.
Restriction analysis was performed to determine the correct
orientation of the insert.
The retrovirus vector carrying the truncated
interleukin-1 receptor is transferred into the CRIP
(Proc. Natl. Acad. Sci., Vol. 85, pp. 6460-6464 (1988),
0. Danos and R. C. Mulligan) packaging cell line using a
standard CaP04 transfection procedure and cells wherein the
viral vector is stably integrated and is selected on the basis
of resistance to the antibiotic G418. The viral vector
cont~ining the neomycin resistant (neo-r) gene is capable of
imparting resistance of the cell line to G418. The CRIP cell
line expresses the three viral proteins required for packaging
the vector viral RNAs into infectious particles. Moreover,
the viral particles produced by the CRIP cell line are able to
efficiently infect a wide variety of mammalian cell types
including human cells. All retroviral particles produced by
this cell line are defective for replication but retain the
ability to stably integrate into synovial cells thereby
becoming an heritable trait of these cells. Virus stocks
produced by this method are substantially free of
cont~in~ting helper-virus particles and are also non-
pathogenic.
More specifically, the truncated interleukin-1 gene
can be inserted into a retroviral vector under the regulation
WO94120517 PCT~S94/0~14
2157782
- 43 -
of a suitable eukaryotic promoter such as the retroviral
promoter already contained within the gene transfer vector,
such as for example, the pLJ vector shown in Figure 9.
Figure 9 shows the structure of the pLJ interleukin receptor
retroviral vector and partial restriction endonuclease map.
Reference numeral lO shows the interleukin-l receptor inserted
into a retroviral vector. Reference numeral 12 indicates long
terminal repeats (LTR's) at eac~ end of the structure of the
pLJ interleukin receptor retroviral vector shown in Figure 8.
These LTR's regulate the viral transcription and expression of
the interleukin-l receptor. Bacterial gene encoding
resistance to the antibiotic neomycin (neo-r) is shown at
reference numeral 16. The Simian Virus 40 enhancer promoter
(SV 40) is indicated at reference numeral 18, and regulates
the expression of the neo-r gene. Reference numbers 20 and
22, respectively, show the sites wherein the resulting
interleukin receptor fragment is cloned. It will be
understood by those persons skilled in the art that other
vectors containing different eukaryotic promoters may also be
utilized to obtain a generally maximal level of interleukin-l
receptor expression. The vectors containing the truncated,
and modified interleukin-l receptor will be introduced into a
retroviral packaging cell line (CRIP) by transfection and
stable transformants isolated by selection for the expression
of the neomycin resistance gene also carried by the pLJ
vector. The CRIP cell line expresses all the proteins
required for packaging of the exogenous retroviral RNA. Viral
particles produced by the G418-selected CRIP cell lines wi l
carry a recombinant retrovirus able to infect mammalian cells
and stably express the interleukin-l truncated receptor. The
viral particles are used to infect synovial cells directly in
vivo by injecting the virus into the ~oint space.
Another embodiment of this invention provides a
method for using the hereinbefore described viral particles to
WO94/20517 PCT~S94/0~14 ~
2~S7~ 8~
-
- 44 -
infect in culture synovial cells obtained from the lining of
the joint of a mammalian host. The advantage of the infection
of synovial cells in culture is that infected cells harboring
the interleukin-l receptor retroviral construct can be
5 selected using G418 for expression of the neomycin resistance
gene. The infected synovial c~lls expressing the interleukin-
1 receptor can then be transplanted back into the joint by
intra-articular injection. The transplanted cells will
express high levels of soluble interleukin-1 receptor in the
joint space thereby binding to and neutralizing substantially
all isoforms of interleukin-l, including interleukin-1 alpha
and interleukin-l beta.
The method used for transplantation of the synovial
cells within the joint is a routine and relatively minor
procedure used in the treatment of chronic inflammatory joint
disease. Although synovium can be recovered from the joint
during open surgery, it is now common to perform
synovectomies, especially of the knee, through the
arthroscope. The arthroscope is a small, hollow rod inserted
into the knee via a small puncture wound. In addition to
permitting the intra-articular insertion of a fibre-option
system, the arthroscope allows access to surgical instruments,
such that snyovial tissue can be removed arthroscopically.
Such procedures can be carried out under "spinal" anesthetic
and the patient allowed home the same day. In this manner
sufficient synovium can be obtained from patients who will
receive this gene therapy.
The synovial cells (synoviocytes) contained within
the excised tissue may be aseptically recovered by enzymic
digestion of the connective tissue matrix. Generally, the
synovium is cut into pieces of approximately 1 millimeter
diameter and digested sequentially with trypsin (0.2% w/v in
Grey's Balanced Salt Solution) for 30 minutes at 37
Centigrade, and collagenase (0.2% w/v in Grey's Balanced Salt
.
WO94/20517 PCT~S94/0~14
2157~82
Solution) for 2 hours at 37 Centigrade. Cells recovered from
this digestion are seeded into plastic culture dishes at a
concentration of 104 - 105 cells per square centimeter with
Hank's F12 medium supplemented with 10% fetal bovine serum and
antibiotics After 3-7 days, the culture medium is withdrawn.
Non-adherent cells such as lymphocytes are removed by washing
with Gey's Balanced Salt Solution and fresh medium added. The
adherent cells can now be used as they are, allowed to grow to
confluency or taken through one or more subcultures.
Subcultivating expands the cell number and removes non-
dividing cells such as macrophages.
Following genetic manipulation of the cells thus
recovered, they can be removed from the culture dish by
trypsinizing, scraping or other m~ans, and made into a
st~n~rd suspension. Gey's Balanced Salt Solution or other
isotonic salt solutions of suitable composition, or saline
solution are suitable carriers. A suspension of cells can
then be injected into the recipient mammalian joint.
Intra-articular injections of this type are routine and easily
carried out in the doctor's office. No surgery is necessary.
Very large numbers of cells can be introduced in this way and
repeat injections carried out as needed.
Another embodiment of this invention is the gene
produced by the hereinbefore described method of preparation.
This gene carried by the retrovirus may be incorporated in a
suitable pharmaceutical carrier, such as for example, buffered
physiologic saline, for parenteral administration. This gene
may be administered to a patient in a therapeutically
effective dose. More specifically, this gene may be
incorporated in a suitable pharmaceutical carrier at a
therapeutically effective dose and administered by
intra-articular injection.
In another embodiment of this invention, this gene
may be administered to patients as a prophylactic measure to
WO94/20517 PCT~S94/0~14 ~
215~78~
- 46 -
prevent the development of arthritis in those patients
determined to be highly susceptible of developing this
disease. More speçifically, this gene carried by the
retrovirus may be incorporated in a suitable pharmaceutical
carrier at a prophylactically effective dose and administered
by parenteral injection, including intra-articular injection.
EXAMPLE X
Fifty mi~L Gy r ams of a DNA plasmid vector molecule
containing the interleukin-l beta coding sequence ligated
downstream of the CMV promoter was encapsulated within
cationic liposomes, mixed with Geys biological buffer and
injected intra-articularly into the knee joints of a rabbit.
Fourty eight hours subsequent to injection one nanogram of
interleukin-1 beta was recovered from the knee joint area.
Therefore, injection of the DNA cont~i n; ng liposome solution
within the region of the synovial tissue prompted fusion of
the liposomes to the synovial cells, transfer of the DNA
plasmid vector into synovial cells and subsequent expression
of the IL-1 beta gene. Additionally, it is possible to inject
non-encapsulated (i.e., naked) DNA into the joint area and
monitor transfection of the DNA vector into the synovial cells
as determined by subsequent expression of the IL-1 beta gene
in synovial cells. Therefore, either method may be utilized
as a plausible alternative to the n vitro manipulation of
synovia also exemplified in the present invention.
It will be appreciated by those skilled in the art
that this invention provides a method of introducing into a
connective tissue cell of a mammalian host n vitro, or in the
alternative in vivo, at least one gene which codes for
proteins with therapeutic properties. This method includes
employing genes having DNA that is capable of maintenance and
expression.
It will be appreciated by those skilled in the art
that this invention provides a method of introducing at least
WO94/20517 215 7 7 8 2 PCT~S94/0~14
- 47 -
one gene encoding a product into at least one cell of the
connective tissue of a mammalian host for treating an
arthritic condition of the mammalian host.
It will be understood by those skilled in the art
that this invention provides a method to repair and regenerate
the connective tissue of a mammalian host.
It will be further understood that this invention
provides a method to produce an animal model for the study of
connective tissue pathology.
It will be appreciated by those persons skilled in
the art that this invention provides a method of using and a
method of preparing a gene encoding an extra cellular
interleukin-l binding domain of an interleukin-l receptor that
is capable of binding to and neutralizing substantially all
isoforms of interleukin-l, and thus substantially protect
cartilage of a mammalian host from pathological degradation.
In addition, it will be understood by those persons skilled in
the art that the method of using the gene of this invention
will reduce inflammation, protect soft tissues of the joint
and suppress the loss of bone that occurs in patients
suffering with arthritis.
It will be appreciated by those persons skilled in
the art that the viral vectors employed in the hereinbefore
described invention may be employed to transfect synovial
cells in vivo or in culture, such as by direct intra-articular
injection or transplantation of autologous synovial cells from
the patient transduced with the retroviral vector carrying the
truncated interleukin-l receptor gene.
Whereas particular embodiments of this invention
have been described above for purposes of illustration, it
will be evident to those persons skilled in the art that
numerous variations of the details of the present invention
may be made without departing from the invention as defined in
the appended claims.
-
WO94/20517 PCT~S94/0~14
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SEQUENCE LISTING
(1) GENERAL INFORMATION: ~
(i) APPLICANT: University of Pittsburgh of the Commonwealth
System of Higher Education
(ii) TITLE OF INVENTION: Gene Transfer For Treating a
Connective Tissue of a Mammalian Host
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Eckert Seamans Cherin ~ Mellott
(B) STREET: 1700 Market Street Suite 3232
(C) CITY: Philadelphia
(D) STATE: PA
(E) COUNTRY: USA
(F) ZIP: 19103
(v) COM~Ul~ READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) CO~U'l'~K: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version ~1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gould, Jr., Lewis F.
(B) REGISTRATION NUMBER: 25,057
(C) REFERENCE/DOCKET NUMBER: 109070-9
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (215) 575-6000
(B) TELEFAX: (215) 575-6015
(C) TELEX: 866172
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1770 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
WO94/20517 . PCT~S94/0~14
~ 2I57782
- 49 -
(ii) MOLECULE TYPE: cDNA
(iii) HYPG~ CAL: NO
- (iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Human T-cell cDNA Library
(B) CLONE: Human Interleukin-1 Receptor
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 55..1764
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
C~lC~lGAGA AGCTGGACCC CTTGGTAAAA GACAAGGCCT TCTC~AA~-AA GAAT ATG 57
Met
AAA GTG TTA CTC AGA CTT ATT TGT TTC ATA GCT CTA CTG ATT TCT TCT lOS
Ly~ Val Leu Leu Arg Leu Ile Cy~ Phe Ile Ala Leu Leu Ile Ser Ser
5 lO 15
CTG GAG GCT GAT AAA TGC AAG GAA CGT GAA GAA AAA ATA ATT TTA GTG 153
Leu Glu Ala Asp Lys Cys Ly~ Glu Arg Glu Glu Lys Ile Ile Leu Val
20 25 30
TCA TCT GCA AAT GAA ATT GAT GTT CGT CCC TGT CCT CTT AAC CCA AAT 201
Ser Ser Ala A~n Glu Ile Asp Val Arg Pro Cys Pro Leu Asn Pro Asn
35 40 45
GAA CAC AAA GGC ACT ATA ACT TGG TAT AAA GAT GAC AGC AAG ACA CCT 249
Glu His Lys Gly Thr Ile Thr Trp Tyr Lys A~p Asp Ser Lys Thr Pro
50 55 60 65
GTA TCT ACA GAA CAA GCC TCC AGG ATT CAT CAA CAC AAA GAG AAA CTT 297
Val Ser Thr Glu Gln Ala Ser Arg Ile Hi~ Gln Hi~ LYB Glu LYB Leu
70 75 80
TGG TTT GTT CCT GCT AAG GTG GAG GAT TCA GGA CAT TAC TAT TGC GTG 345
Trp Phe Val Pro Ala Lys Val Glu A8p Ser Gly Hi~ Tyr Tyr Cys Val
85 90 95
GTA AGA AAT TCA TCT TAC TGC CTC AGA ATT AAA ATA AGT GCA AAA TTT 393
Val Arg Asn Ser Ser Tyr CYB Leu Arg Ile Lys Ile Ser Ala Lys Phe
100 105 110
GTG GAG AAT GAG CCT AAC TTA TGT TAT AAT GCA CAA GCC ATA TTT AAG 441
Val Glu A~n Glu Pro Asn Leu Cy8 Tyr A~n Ala Gln Ala Ile Phe Lys
115 120 125
CAG AAA CTA CCC GTT GCA GGA GAC GGA GGA CTT GTG TGC CCT TAT ATG 489
Gln Lys Leu Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr Met
130 135 140 145
-
WO 94120517 . PCT/US94/02414
2~1$~
- 50 -
GAG TTT TTT AAA AAT GAA A~T AAT GAG TTA CCT AaA TTA CAG TGG TAT 537
Glu Phe Phe Lys Asn Glu A~n A~n Glu Leu Pro Ly~ Leu Gln Trp Tyr
150 -155 160
AAG GAT TGC AAA CCT CTA CTT CTT GAC AAT ATA CAC TTT AGT GGA GTC 585
Lys Asp Cy~ Ly~ Pro Leu Leu Leu Asp Asn Ile Hi~ Phe Ser Gly Val
165 170 175
AAA GAT AGG CTC ATC GTG ATG AAT GTG GCT GAA AAG CAT AGA GGG AAC 633
Lys Asp Arg Leu Ile Val Met Asn Val Ala Glu Ly~ His Arg Gly Asn
180 185 190
TAT ACT TGT CAT GCA TCC TAC ACA TAC TTG GGC AAG CAA TAT CCT ATT 681
Tyr Thr Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro Ile
195 200 205
ACC CGG GTA ATA GAA TTT ATT ACT CTA GAG GAA AAC AAA CCC ACA AGG 729
Thr Arg V~l Ile Glu Phe Ile Thr Leu Glu Glu Asn Ly~ Pro Thr Arg
210 215 220 225
CCT GTG ATT GTG AGC CCA GCT AAT GAG ACA ATG GAA GTA GAC TTG GGA 777
Pro Val Ile Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu Gly
230 235 240
TCC CAG ATA CAA TTG ATC TGT AAT GTC ACC GGC CAG TTG AGT GAC ATT 825
Ser Gln Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp Ile
245 250 255
GCT TAC TGG AAG TGG AAT GGG TCA GTA ATT GAT GAA GAT GAC CCA GTG 873
Ala Tyr Trp Ly~ Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro Val
260 265 270
CTA GGG GAA GAC TAT TAC AGT GTG GAA AAT CCT GCA AAC AAA AGA AGG 921
Leu Gly Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Ly~ Arg Arg
275 280 285
AGT ACC CTC ATC ACA GTG CTT AAT ATA TCG GA~ ATT GAA AGT AGA TTT 969
Ser Thr Leu Ile Thr Val Leu Asn Ile Ser Glu Ile Glu Ser Arg Phe
290 295 300 305
TAT AAA CAT CCA TTT ACC TGT TTT GCC AAG AAT ACA CAT GGT ATA GAT 1017
Tyr Lys His Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly Ile Asp
310 315 320
GCA GCA TAT ATC CAG TTA ATA TAT CCA GTC ACT AAT TTC CAG AAG CAC 1065
Ala Ala Tyr Ile Gln Leu Ile Tyr Pro Val Thr Asn Phe Gln Ly~ His
325 330 335
ATG ATT GGT ATA TGT GTC ACG TTG ACA GTC ATA ATT GTG TGT TCT GTT 1113
Met Ile Gly Ile Cys Val Thr Leu Thr Val Ile Ile Val Cys Ser Val
340 345 350
TTC ATC TAT AAA ATC TTC A~G ATT GAC ATT GTG CTT TGG TAC AGG GAT 1161
Phe Ile Tyr Lys Ile Phe Lys Ile Asp Ile Val Leu Trp Tyr Arg Asp
355 360 365
TCC TGC TAT GAT TTT CTC CCA ATA AAA GCT TCA GAT GGA AAG ACC TAT 1209
Ser Cys Tyr Asp Phe Leu Pro Ile Lys Ala Ser Asp Gly Ly~ Thr Tyr
370 375 380 385
GAC GCA TAT ATA CTG TAT CCA AAG ACT GTT GGG GAA GGG TCT ACC TCT 1257
-
WO94120517 PCT~S94/0~14
~ 2157782
- 51 -
A~p Ala Tyr Ile Leu Tyr Pro Lys Thr Val Gly Glu Gly Ser Thr Ser
390 395 400
GAC TGT GAT ATT ~TT GTG TTT AAA GTC TTG CCT GAG GTC TTG GAA AAA 1305
Agp Cy8 Asp Ile Phe Val Phe Lys Val Leu Pro Glu Val Leu Glu Lys
405 410 415
CAG TGT GGA TAT AAG CTG TTC ATT TAT GGA AGG GAT GAC TAC GTT GGG 1353
Gln Cy~ Gly Tyr Lys Leu Phe Ile Tyr Gly Arg Asp Asp Tyr Val Gly
420 425 430
GAA GAC ATT GTT GAG GTC ATT AAT GAA AAC GTA AAG AAA AGC AGA AGA 1401
Glu Asp Ile Val Glu Val Ile Asn Glu A~n Val Ly~ Ly~ Ser Arg Arg
435 440 445
CTG ATT ATC ATT TTA GTC AGA GAA ACA TCA GGC TTC AGC TGG CTG GGT 1449
Leu Ile Ile Ile Leu Val Arg Glu Thr Ser Gly Phe Ser Trp Leu Gly
450 455 460 465
GGT TCA TCT GAA GAG CAA ATA GCC ATG TAT AAT GCT CTT GTT CAG GAT 1497
Gly Ser Ser Glu Glu Gln Ile Ala Met Tyr A~n Ala Leu Val Gln Asp
470 475 480
GGA ATT AAA GTT GTC CTG CTT GAG CTG GAG AAA ATC CAA GAC TAT GAG 1545
Gly Ile Lys Val Val Leu Leu Glu Leu Glu Ly~ Ile Gln A~p Tyr Glu
485 490 495
AAA ATG CCA GAA TCG ATT AAA TTC ATT AAG CAG AAA CAT GGG GCT ATC 1593
Ly~ Met Pro Glu Ser Ile Ly~ Phe Ile Ly~ Gln Lys His Gly Ala Ile
500 505 510
CGC TGG TCA GGG GAC TTT ACA CAG GGA CCA CAG TCT GCA AAG ACA AGG 1641
Arg Trp Ser Gly Asp Phe Thr Gln Gly Pro Gln Ser Ala Lys Thr Arg
515 520 525
TTC TGG AAG AAT GTC AGG TAC CAC ATG CCA GTC CAG CGA CGG TCA CCT 1689
Phe Trp Lys A~n Val Arg Tyr His Met Pro Val Gln Arg Arg Ser Pro
530 535 540 545
TCA TCT AAA CAC CAG TTA CTG TCA CCA GCC ACT AAG GAG AAA CTG CAA 1737
Ser Ser Ly~ His Gln Leu Leu Ser Pro Ala Thr Ly~ Glu Ly~ Leu Gln
550 555 560
AGA GAG GCT CAC GTG CCT CTC GGG TAGCATGGA 1770
Arg Glu Ala His Val Pro Leu Gly
565 570
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 569 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
WO 94/20517 PCT/US94/02414 ~
~,~5~
-- 52 ~
Met Lys Val Leu Leu Arg Leu Ile Cys Phe Ile Ala Leu Leu Ile Ser
.10 15
Ser Leu Glu Ala A~p Lys Cys L-ys Glu Arg Glu Glu Lys I le I le Leu
Val Ser Ser Ala Asn Glu Ile Asp Val Arg Pro CYD Pro Leu Asn Pro
Asn Glu His Lys Gly Thr Ile Thr Trp Tyr Lys Asp Asp Ser Ly~ Thr
Pro Val Ser Thr Glu Gln Ala Ser Arg Ile His Gln His Lys Glu Lys
Leu Trp Phe Val Pro Ala Lys Val Glu Asp Ser Gly His Tyr Tyr Cys
Val Val Arg Asn Ser Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys
100 105 110
Phe Val Glu Asn Glu Pro Asn Leu Cys Tyr Asn Ala Gln Ala I le Phe
115 120 125
Lys Gln Lys Leu Pro Val Ala Gly Asp Gly Gly Leu Val Cys Pro Tyr
130 135 140
Met Glu Phe Phe Lys Asn Glu Asn Asn Glu Leu Pro Lys Leu Gln Trp
145 150 155 160
Tyr Lys Asp Cys Lys Pro Leu Leu Leu A~p Asn Ile His Phe Ser Gly
165 170 175
Val Lys Asp Arg Leu Ile Val Met Asn Val Ala Glu Ly~ His Arg Gly
180 185 190
Asn Tyr Thr Cys His Ala Ser Tyr Thr Tyr Leu Gly Lys Gln Tyr Pro
195 200 205
Ile Thr Arg Val Ile Glu Phe Ile Thr Leu Glu Glu Asn Lys Pro Thr
210 215 220
Arg Pro Val Ile Val Ser Pro Ala Asn Glu Thr Met Glu Val Asp Leu
225 230 235 240
Gly Ser Gln Ile Gln Leu Ile Cys Asn Val Thr Gly Gln Leu Ser Asp
245 250 255
Ile Ala Tyr Trp Lys Trp Asn Gly Ser Val Ile Asp Glu Asp Asp Pro
260 265 270
Val Leu Gly Glu Asp Tyr Tyr Ser Val Glu Asn Pro Ala Asn Lys Arg
275 280 285
Arg Ser Thr Leu Ile Thr Val Leu Afin Ile Ser Glu Ile Glu Ser Arg
290 295 300
Phe Tyr Lys His Pro Phe Thr Cys Phe Ala Lys Asn Thr His Gly Ile
305 310 315 320
Asp Ala Ala Tyr Ile Gln Leu Ile Tyr Pro Val Thr Asn Phe Gln Lys
W094n0517 . PCT~S94/0~14
- 2157782
- 53 -
325 330 335
His Met Ile Gly Ile Cy~ Val Thr Leu Thr Val Ile Ile Val Cys Ser
340 345 350
Val Phe Ile Tyr Lys Ile Phe Ly~ Ile ARP Ile Val Leu Trp Tyr Arg
355 360 365
Asp Ser Cy~ Tyr Asp Phe Leu Pro Ile Ly~ Ala Ser Asp Gly Lys Thr
370 375 380
Tyr A~p Ala Tyr Ile Leu Tyr Pro Lys Thr Val Gly Glu Gly Ser Thr
385 390 395 400
Ser Asp Cys Asp Ile Phe Val Phe Lys Val Leu Pro Glu Val Leu Glu
405 410 . 415
Lys Gln Cys Gly Tyr Lys Leu Phe Ile Tyr Gly Arg Asp Asp Tyr Val
420 425 430
Gly Glu Asp Ile Val Glu Val Ile Asn Glu Asn Val Lys Lys Ser Arg
435 440 445
Arg Leu Ile Ile Ile Leu Val Arg Glu Thr Ser Gly Phe Ser Trp Leu
450 455 460
Gly Giy Ser Ser Glu Glu Gln Ile Ala Met Tyr A~n Ala Leu Val Gln
465 470 475 480
Asp Gly Ile Ly~ Val Val Leu Leu Glu Leu Glu Lys Ile Gln Asp Tyr
485 490 495
Glu Ly~ Met Pro Glu Ser Ile Ly~ Phe Ile Lys Gln Lys His Gly Ala
500 505 510
Ile Arg Trp Ser Gly A~p Phe Thr Gln Gly Pro Gln Ser Ala Lys Thr
515 520 525
Arg Phe Trp Lys Asn Val Arg Tyr His Met Pro Val Gln Arg Arg Ser
530 535 540
Pro Ser Ser Lys His C-ln Leu Leu Ser Pro Ala Thr Lys Glu Ly~ Leu
545 550 555 560
Gln Arg Glu Ala His Val Pro Leu Gly
565
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 178~ base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
WOg4/20517 . PCT~S94/0~14 ~
2~,S~Ss~ ' '
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(A) LIBRARY: Mouse T-cell cDNA Library
(B) CLONE: ~ouse Interleukin-1 Receptor
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 46..1776
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GGATGTCATC AGAGTTCCCA GTGCCCC~-~A CCGTGAACAA CACAA ATG GAG AAT 54
Met Glu Asn
ATG AAA GTG CTA CTG GGG CTC ATT TGT CTC ATG GTG CCT CTG CTG TCG 102
Met Lys Val Leu Leu Gly Leu Ile Cys Leu Met Val Pro Leu Leu Ser
5 10 15
CTG GAG ATT GAC GTA TGT ACA GAA TAT CCA AAT CAG ATC GTT TTG TTT 150
Leu Glu Ile Asp Val Cys Thr Glu Tyr Pro Asn Gln Ile Val Leu Phe
20 25 30 35
TTA TCT GTA AAT GAA ATT GAT ATT CGC AA5 TGT CCT CTT ACT CCA AAT 198
Leu Ser Val Asn Glu Ile Asp Ile Arg Lys Cys Pro Leu Thr Pro Asn
40 45 50
AAA ATG CAC GGC GAC ACC ATA ATT TGG TAC AAG AAT GAC AGC AAG ACC 246
LYB Met His Gly Asp Thr ILe Ile Trp Tyr Ly~ Asn Asp Ser Lys Thr
55 60 65
CCC ATA TCA GCG GAC CGG GAC TCC AGG ATT CAT CAG CAG AAT GAA CAT 294
Pro Ile Ser Ala Asp Arg Asp Ser Arg Ile His Gln Gln Asn Glu His
70 75 80
CTT TGG TTT GTA CCT GCC AAG GTG GAG GAC TCA GGA TAT TAC TAT TGT 342
Leu Trp Phe Val Pro Ala Ly~ Val Glu Asp Ser Gly Tyr Tyr Tyr Cys
85 90 95
ATA GTA AGA AAC TCA ACT TAC TGC CTC AAA ACT AAA GTA ACC GTA ACT 390
Ile Val Arg Asn Ser Thr Tyr Cys Leu Lys Thr Lys Val Thr Val Thr
100 105 110 115
GTG TTA GAG A~T GAC CCT GGC TTG TGT TAC AGC ACA CAG GCC ACC TTC 438
Val Leu Glu Asn Asp Pro Gly Leu Cys Tyr Ser Thr Gln Ala Thr Phe
120 125 130
CCA CAG CGG CTC CAC ATT GCC GGG GAT GGA AGT CTT GTG TGC CCT TAT 486
Pro Gln Arg Leu His Ile Ala Gly Asp Gly Ser Leu Val Cys Pro Tyr
135 140 145
GTG AGT TAT TTT AAA GAT GAA AAT AAT GAG TTA CCC GAG GTC CAG TGG 534
Val Ser Tyr Phe Lys Asp Glu Asn Asn Glu Leu Pro Glu Val Gln Trp
150 155 160
TAT AAG AAC TGT AAA CCT CTG CTT CTT GAC AAC GTG AGC TTC TTC GGA 582
WO 94/20517 PCT/US94/02414
-- 2157~82
-- 55 ~
Tyr LYB A~n Cy~ Lys Pro Leu Leu Leu A~p Ann Val Ser Phe Phe Gly
165 170 175
GTA AAA GAT AP~A CTG TTG GTG AGG AAT GTG GCT GAA GAG CAC AGA GGG 630
Val LYB ABP LYB Leu Leu Val Arg Asn Val Ala Glu Glu His Arq Gly
180 185 190 195
GAC TAT ATA TGC CGT ATG TCC TAT ACG TTC CGG GGG AAG CAA TAT CCG 678
Asp Tyr Ile Cys Arg Met Ser Tyr Thr Phe Arg Gly Lys Gln Tyr Pro
200 205 210
GTC ACA CGA GTA ATA CAA TTT ATC ACA ATA GAT GAA AAC AAG AGG GAC 726
Val Thr Arg Val Ile Gln Phe Ile Thr Ile ABP Glu AYn Lys Arg Asp
215 220 225
AGA CCT GTT ATC CTG AGC CCT CGG AAT GAG ACG ATC GAA GCT GAC CCA 7?4
Arg Pro Val Ile Leu Ser Pro Arg Asn Glu Thr Ile Glu Ala Asp Pro
230 235 240
GGA TCA ATG ATA CAA CTG ATC TGC AAC GTC ACG GGC CAG TTC TCA GAC 822
Gly Ser Met Ile Gln Leu Ile Cy8 Asn Val Thr Gly Gln Phe Ser Asp
245 250 255
CTT GTC TAC TGG AAG TGG AAT GGA TCA GAA ATT GAA TGG AAT GAT CCA 870
Leu Val Tyr Trp Ly~ Trp Asn Gly Ser Glu Ile Glu Trp Asn Asp Pro
260 265 270 275
TTT CTA GCT GAA GAC TAT CAA TTT GTG GAA CAT CCT TCA ACC AAA AGA 918
Phe Leu Ala Glu Asp Tyr Gln Phe Val Glu His Pro Ser Thr Lys Arg
280 285 290
AAA TAC ACA CTC ATT ACA ACA CTT AAC ATT TCA GAA GTT AAA AGC CAG 966
Lys Tyr Thr Leu Ile Thr Thr Leu Asn Ile Ser Glu Val Lys Ser Gln
295 300 305
TTT TAT CGC TAT CCG TTT ATC TGT GTT GTT AAG AAC ACA AAT ATT TTT 1014
Phe l'yr Arg Tyr Pro Phe I le Cys Val Val Lys Asn Thr Asn I le Phe
310 315 320
GAG TCG GCG CAT GTG CAG TTA ATA TAC CCA GTC CCT GAC TTC AAG AAT 1062
Glu Ser Ala His Val Gln Leu Ile Tyr Pro Val Pro Asp Phe Ly~ Asn
325 330 335
TAC CTC ATC GGG GGC TTT ATC ATC CTC ACG GCT ACA ATT GTA TGC TGT 1110
Tyr Leu Ile Gly Gly Phe Ile Ile Leu Thr Ala Thr Ile Val Cy~ Cys
340 345 350 355
GTG TGC ATC TAT AP~A GTC TTC AAG GTT GAC ATA GTG CTT TGG TAC AGG 1158
Val Cy~ Ile Tyr Lys Val Phe LYB Val Asp Ile Val Leu Trp Tyr Arg
360 365 370
GAC TCC TGC TCT GGT TTT CTT CCT TCA AAA GCT TCA GAT GGA AAG ACA 1206
Asp Ser Cys Ser Gly Phe Leu Pro Ser Lys Ala Ser Asp Gly Lys Thr
375 380 385
TAC GAT GCA TAT ATT CTT TAT CCC AAG ACC CTG GGA GAG GGG TCC TTC 1254
Tyr Asp Ala Tyr Ile Leu Tyr Pro Lys Thr Leu Gly Glu Gly Ser Phe
390 395 400
TCA GAC TTA GAT ACT TTT GTT TTT A~A CT-', TTG CCT GAG GTC TTG GAG 1302
Ser Asp Leu Asp Thr Phe Val Phe Lys Leu Leu Pro Glu Val Leu Glu
W O 94120517 PCTruS94/024l4 ~
2ls~8~
- 56 -
405 410 415
GGA CAG TTT GGA TAC AAG CTG TTC ATT~TAT GGA AGG GAT GAC TAT GTT 1350
Gly Gln Phe Gly Tyr Ly~ Leu Phe Iie Tyr Gly Arg Asp Asp Tyr Val
420 425 430 435
GGA GAA GAT ACC ATC GAG GTT ACT AAT GAA AAT GTA AAG A~A AGC AGG 1398
Gly Glu A~p Thr Ile Glu Val Thr Asn Glu A~n Val Lys Ly~ Ser Arg
440 445 450
AGG CTG ATT ATC ATT CTA GTG AGA GAT ATG GGA GGC TTC AGC TGG CTG 1446
Arg Leu Ile Ile Ile Leu Val Arg A~p Met Gly Gly Phe Ser Trp Leu
455 460 465
GGC CAG TCA TCT GAA GAG CAA ATA GCC ATA TAC AAT GCT CTC ATC CAG 1494
Gly Gln Ser Ser Glu Glu ~ln Ile Ala Ile Tyr Asn Ala Leu Ile Gln
470 475 480
GAA GGA ATT AAA ATC GTC CTG CTT GAG TTG GAG A~A ATC CAA GAC TAT 1542
Glu Gly Ile Lys Ile Val Leu Leu Glu Leu Glu Ly~ Ile Gln A~p Tyr
485 490 495
GAG AAA ATG CCA GAT TCT ATT CAG TTC ATT AAG CAG AAA CAC GGA GTC 1590
Glu Lys Met Pro Asp Ser Ile Gln Phe Ile Lys Gln Lys His Gly Val
500 505 510 515
ATT TGC TGG TCA GGA GAC TTT CAA GAA AGA CCA CAG TCT GCA AAG ACC 1638
Ile Cy~ Trp Ser Gly Asp Phe Gln Glu Arg Pro Gln Ser Ala Lys Thr
520 525 530
AGG TTC TGG AAA AAC TTA AGA TAC CAG ATG CCA GCC CAA CGG AGA TCA 1686
Arg Phe Trp Ly~ Asn Leu Arg Tyr Gln Met Pro Ala Gln Arg Arg Ser
535 540 545
CCA TTG TCT AAA CAC CGC TTA CTA ACC CTG GAT CCT GTG CGG GAC ACT 1734
Pro Leu Ser Ly~ His Arg Leu Leu Thr Leu Asp Pro Val Arg A~p Thr
550 555 560
AAG GAG A~A CTG CCG GCA GCA ACA CAC TTA CCA CTC GGC TAGCATGGC 1782
Lys Glu Lys Leu Pro Ala Ala Thr His Leu Pro Leu Gly
565 570 575
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQ'JENCE CHARACTERISTICS:
(A) LENGTH: 576 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Glu Asn Met Lys Val Leu Leu Gly Leu Ile Cys Leu Met Val Pro
1 5 10 15
Leu Leu Ser Leu Glu Ile Asp Val Cy8 Thr Glu Tyr Pro Asn Gln Ile
WO 94/20517 . PCT/US94/02414
21S7782
- 57 -
Val Leu Phe Leu Ser Val Asn Glu Ile Asp Ile Arg Lys Cy5 Pro Leu
Thr Pro Asn Lys Met His Gly Asp Thr Ile Ile Trp Tyr Lys A~n A~p
Ser Lys Thr Pro Ile Ser Ala Asp Arq Asp Ser Arg Ile His Gln Gln
A~n Glu His Leu Trp Phe Val Pro Ala Ly~ Val Glu Asp Ser Gly Tyr
Tyr Tyr Cys Ile Val Arg Asn Ser Thr Tyr Cy8 Leu Lys Thr Lys Val
100 105 110
Thr Val Thr Val Leu Glu Asn Asp Pro Gly Leu Cys Tyr Ser Thr Gln
115 120 125
Ala Thr Phe Pro Gln Arg Leu His Ile Ala Gly Asp Gly Ser Leu Val
130 135 140
Cys Pro Tyr Val Ser Tyr Phe Lys Asp Glu Asn Asn Glu Leu Pro Glu
145 150 155 160
Val Gln Trp Tyr Lys Asn Cys Lys Pro Leu Leu Leu Asp Asn Val Ser
165 170 175
Phe Phe Gly Val Lys Asp Lys Leu Leu Val Arg Asn Val Ala Glu Glu
180 185 190
Hi~- Arg Gly Asp Tyr Ile Cys Arg Met Ser Tyr Thr Phe Arg Gly Lys
195 200 205
Gln Tyr Pro Val Thr Arg Val Ile Gln Phe Ile Thr Ile A~p Glu Asn
210 215 220
Lys Arg Asp Arg Pro Val Ile Leu Ser Pro Arg Asn Glu Thr Ile Glu
225 230 235 240
Ala Asp Pro Gly Ser Met Ile Gln Leu Ile Cy5 Asn Val Thr Gly Gln
245 250 255
Phe Ser Asp Leu Val Tyr Trp Lys Trp Asn Gly Ser Glu Ile Glu Trp
260 265 270
Asn Asp Pro Phe Leu Ala Glu Asp Tyr Gln Phe Val Glu His Pro Ser
275 280 285
Thr Lys Arg Ly~ Tyr Thr Leu Ile Thr Thr Leu Asn Ile Ser Glu Val
290 295 300
Lys Ser Gln Phe Tyr Arg Tyr Pro Phe Ile Cys Val Val Lys Asn Thr
305 310 315 320
Asn Ile Phe Glu Ser Ala His Val Gln Leu Ile Tyr Pro Val Pro Asp
325 330 335
Phe Lys Asn Tyr Leu Ile Gly Gly Phe Ile Ile Leu Thr Ala Thr Ile
340 345 350
Val Cys Cys Val Cys Ile Tyr Lys Val Phe Ly~ Val Asp Ile Val Leu
WO94/20517 ~ ~ PCT~S94/0~14
- 58 -
355 360 365
Trp Tyr Arg A~p Ser Cy~ Ser Gly Phe Leu P~o Ser Ly~ Ala Ser Asp
370 375 380
Gly Ly~ Thr Tyr Anp Ala Tyr Il~ ~eu Tyr Pro Lys Thr Leu Gly Glu
385 390 395 400
Gly Ser Phe Ser A~p Leu A3p Thr Phe Val Phe Ly~ Leu Leu Pro Glu
405 410 415
Val Leu Glu Gly Gln Phe Gly Tyr Lys Leu Phe Ile Tyr Gly Arg Asp
420 425 430
Asp Tyr Val Gly Glu Asp Thr Ile Glu Val Thr Asn Glu A6n Val Lys
435 440 445
Ly~ Ser Arg Arg Leu Ile Ile Ile Leu Val Arg Asp Met Gly Gly Phe
450 455 460
Ser Trp Leu Gly Gln Ser Ser Glu Glu Gln Ile Ala Ile Tyr Asn Ala
465 470 475 480
Leu Ile Gln Glu Gly Ile Ly~ Ile Val Leu Leu Glu Leu Glu Lys Ile
485 490 495
Gln A~p Tyr Glu Lys Met Pro Asp Ser Ile Gln Phe Ile Lys Gln Lys
500 505 510
Hi~ Gly Val Ile Cys Trp Ser Gly A6p Phe Gln Glu Arg Pro Gln Ser
515 520 525
Ala Ly~ Thr Arq Phe Trp Ly~ A~n Leu Arg Tyr Gln Met Pro Ala Gln
530 535 540
Arg Arg Ser Pro Leu Ser Lys His Arg Leu Leu Thr Leu Asp Pro Val
545 550 555 560
Arg Asp Thr Lys Glu Lys Leu Pro Ala Ala Thr His Leu Pro Leu Gly
565 570 575
(2) INFORMATION FO~ SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Primer oligonucleotide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
WO94120517 PCT~Ss4/0~14
21577$2
- 59 -
(B) CLONE: Primer Oligonuleotide to 5' Leader
Sequence of IL-~ Receptor
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
GCGGATCCCC TCCTGAGAAG CT 22
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2l base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY. linear
(ii) MOLECULE TYPE: Primer oligonucleotide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vii) IMMEDIATE SOURCE:
(B) CLONE: Primer Oligonucleotide Upstream of
Transmembrane Portion of IL-l Receptor
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
GCGGATCCCA TGTGCTACTG G 21
