Note: Descriptions are shown in the official language in which they were submitted.
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1
This invention was made with government support
under grant numbers DK 43727, DK 47754 and DK 506H6 awarded
by the National Institutes of Health. The United States
Government has certain rights in this invention.
This invention relates to generally to methods of
gene delivery and, more specifically to cell implantation
methods for sustained engraftment and long term gene
expression.
The availability of recombinant cloning and
expression methods has provided significant advances in the
therapeutic treatment c7f human diseases. For example, the
availability of recombinant human erythropoietin (Epo), a
regulator of red blood cell production and maintenance, has
provided a significant. advance ire the treatment of renal
failure patients receiving dialysis with the elimination of
attendant dangers of transfusion therapy and an increase in
the quality of life of these patients. The administration
of recombinant Epo is now widely used for long-term
treatment of anemia associated with chronic renal failure,
cancer chemotherapy, and human i..mmunodeficiency virus
infections. Long-term, sustained delivery of this hormone,
and others like it, by cell therapy :rather than by repeated
injections would provide substantial. clinical and economic
benefits. Unfortunately t:he DNA regulatory sequences which
control the expression of Epo in response to tissue
oxygenation are toa large to be suitable for insertion in
a retro viral vector. Similar problems exist Lor the
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2
expression of other hormones and therapeutic proteins as
well.
Long-term in vivo gene expression requires both
target cells and gene delivery vectors that permit
continuous vector encoded activity. of the three prevalent
virus-based methods of gene transfer, retro viral vectors
are likely the most useful for ex vivo gene transfer.
Adeno-associated virus(AAV) vectors have some attractive
features, such as safety and abil.i.ty to transduce non-
proliferating ce:Lls brat do not possess advantages over
retro viruses for ex-vivo gene transduc~ion. Replication
defective retro viral vectors can be made with high titers,
will infect a wide variety of cell. types and infection
results in stable proviral integration into the host
chromosome providing gene expression for the lifetime of
the cell and its progeny. Therapeutic genes can be
expressed at high level from the viral long terminal repeat
(LTR) promoter/enhancer o r strong internal promoters and
the incorporation of internal ribosome entry sites from
picornaviruses into retro viral vectors has allowed the
generation of bicistronic vectors conferring linked-gene
selection. However, there is always great concern when
using replication defective vectors because contamination
with a single replication compentant vector can result in
infection of the patient with a disease producing virus.
Non-hematopoietic cells studied as alternative
vehicles for gene therapy include skin fibroblasts,
myoblasts and vascular smooth muscle cells. Skin
fibroblasts are easily obtained, cultured and transduced
but have a major disadvantage oi~ inactivating vector
sequences after transplantation. Myoblasts represent one
target cell type for gene therapy. Transduced skeletal
myoblasts have been uaed to de:L.i ver Epo in mice and to
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3
treat alpha-L-iduronidase deficiency in dogs following
transplantation. Additionally, intramuscular injection of
plasmid DNA has produced systemic expression of Epo and
other therapeutic proteins in mice.
Smooth muscle cells are present within the
vasculature as a multilayered mass of long-lived cells in
proximity to the circulation and also have been
investigated as targets for gene therapy. Moreover,
transduced vascular smooth muscle cells seeded into carotid
arteries in a rat model has provide sustained expression of
both marker and therapeutic genes, including for example
Epo and granulocyte-colony stimulating factor (C-CSF).
However, this procedure is significantly hindered far human
therapeutic applications because it requires arterial
injury to achieve cell engraftment.
Thus, there exists a need for a reliable and
reproducible method which provides long-term sustained gene
expression of a therapeutic or beneficial protein with
minimal adverse side effects. The present invention
satisfies this need and provides related advantages as
well.
The invention provides a method of systemic
delivery of a gene product t.o an individual. The method
consists of implanting within the wall of the digestive
tract of an individual a prosthetic chamber containing an
effective amount of cells secreting the gene product.
The prosthetic chamber ran be implanted within or
proximal to a connective tissue layer including, for
example, between the tunics serosa and the tunics
muscularis of the digestive tract wall. The gene product
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to be delivered can be selected from cytokines, hormones,
growth factors and bland clotting factors including, for
example, Epo or insulin. Expression of the gene product
can be constitutive or inducible. Therefore, the
invention provides a method of treating a pathology
mediated by the deficiency of a gene product. The method
consists of implanting a prosthetic chamber containing an
effective amount of cells expressing therapeutic levels
of the deficient gene product.
~IRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schemati~~ cross section of a
implantation site under t:he tunica serosa of the stomach.
Figure 2 shows the hematocrit values of animals
implanted with Epo-secreting, LrEpSN-transduced (closed
symbols), and ADA-scret:ing, hASN-transduced (open
symbols , vascular smooth muscle cells as a function of
time.
Figure 3 shows histological cross-sections of
PTFE chambers containing transduced smooth muscle cells
implanted in the stomach.
Figure 9 shows expression levels of endogenous
Epo mRNA in animals rece:i.ving Epo-secreting cell implants
compared to ADA-secreting control cell implants.
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This invention is directed to cell
transplantation methods for the long-term, sustained
delivery of a therapeutic or beneficial protein product.
5 The methods of the invention employ a biocompatable
prosthetic chamber surgically implanted into an
individual and seeded with the cells expressing a
therapeutic or beneficial gene product. The implanted
cells become engrafted, providing consistent, long-term
expression of the therapeutic product. The method is
safe, enables easy engraftment and removal of implanted
smooth muscle cells, and can be applied generally to the
systemic delivery of a variety of therapeutic proteins
including, for example, cytakines, hormones, enzymes and
clotting factors.
In one embodiment, the prosthetic chamber is
implanted at the tissue plane between the tunica serosa
and the tunica muscularis which provides a rich growth
environment for retention of transduced cells.
Therefore, one advantage of the method is that this
tissue is composed of smooth muscle cells, is well
vascularized and able to provide nutrition for implanted
cells with concommitant secretion of the gene product
into the circulatory system.
In another embodiment, the methods of the
invention implant under the tunica serosa genetically
modified vascular smooth muscle cells for stable
long-term cell engraftment expression of Epo. Smooth
muscle cells are present at this location and
advantageously potentiate the engraftment efficiency and
long-term survival of the transplanted cells because
they are a normal constituent of the implanted area. In
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this embodiment, autologous transduced cells are injected
into a chamber created from a polytetrafluaroethylene
(PTFE) ring placed between the serosa and muscularis of
the stomach. The engrafted PTFE ring becomes
vascularized and permits both the long-term survival of
transduced smooth muscle cells and an area protected from
ingrowth of non-transduced cells.
As used herein, the term "systemic" when used
in reference to expressian of a gene product is intended
to mean delivery of the gene product to the circulatory
system of an individual. The circulatary system includes
those parts of the body receiving blood through either
the aorta or the pulmonary artery.
As used herein, the term "delivery" is intended
to mean presenting to the hematopoietic system the
expressed gene product.. Presentation ~~an include, for
example, direct secreta.on into the circulatory system or
indirect secretion into the extracellular space such that
diffusion into capillary beds occurs. Presentation also
can include surface expression an the plasma membrane of
the implanted cells where cells oz t:he hematopoieti.c
system can come in contact with the expressed gene
product. Specific examples of ser_reted gene products
include direct or indirect secretion of cytokines,
hormones, growth factors, extracellular matrix proteins
and vaccine antigens. Delivery of cell. surface proteins
can include, for example:, cell vaccines which express
tumor antigens. Those skilled in the art will know which
mode of delivery is applicable to achieve a particular
result for a particular gene product.
As used herein, the term "gene product" is
intended to mean both polypeptide and nucleic acid
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products that can be expressed or replicated from a
nucleic acid. Specifi~~ examples of a polypeptide gene
product includes peptides, polypeptides or proteins
encoded by structural genes. Specific examples of a
nucleic acid gene product includes RNA such as mRNA,
antisense RNA or DNA and viral vectors for example.
As used herein, the term "'implanting" is
intended to mean the introduction or transplantation of
cells in a prosthetic ~~hamber into an individual wherein
the cells remain viable after implantation and maintain
their gene product expression capability. Implanting
includes, for example, directly introducing the cell-
containing prosthetic ~~hamber into a tissue, introduction
of the cell-containing prosthetic chamber with accessory
cells which are capable of grafting to the surrounding
tissue or which facilate long-term stability of the
prosthetic chamber or gene product expression capability
of the implanted cells, Such accessory cells can be, for
example, Located within the prosthetic chamber, attached
to, or, associated with the perimeter of the chamber.
Implanting additionally includes, for example,
introduction of the cell-containing prasthetic chamber
into the tissue with other components such as
extracellular matrix components, fragments or other
molecules which facilitate adhesion of the cells as well
as with soluble or substrate bound growth factors, for
example. Implanting is generally performed by surgical
procedures but other methods can similarly be employed so
long as the manipulation is compatible for use with the
particular type of prosthetic chamber. being used in to
implant the cells. Those s killed in the art will know,
or can determine, which mode of introduction is
appropriate for a particular type of chamber.
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As used herein, the term "prosthetic chamber"
is intended to mean a devise that is capable of
compartmentalizing cells. Compartmentalization allows
the initial separation of implanted cells from endogenous
cells within the implanted area without substantially
affecting the exchange of nutrients,. cell by-products and
factors necessary for cell viability and proliferation.
The compartmentalization effects performed by the
prosthetic chamber should also allow the cells to
maintain their gene product expression capability.
Barriers which form the compartment can be temporary or
permanent and can effect the complet=a encasement of the
cells, or alternatively, can allow growth of the
implanted cells over and around the barriers as the cells
become engrafted and enlarged in the area of
implantation. Temporary barriers can include, for
example, biodegradable material whereas permanent
barriers can include, far example, metals such as
titanium and stainless stee.l., synthetic polymers and
biopolymer. The sizes of such protheti.c chambers can
vary depending on the number. of cells t:o be transplanted
and can be about lOmm or greater, between about 7-l.Omm
and preferably between about 3-6mm in outer diameter or
width. However, sizes larger or smaller can be used
which can accommodate a desired number of cells or which
is suitable for a particular locatian for implantation.
Those skilled in the art will know what sizes within the
above range of sizes, as well as prosthetic chambers that
lager or smaller than these sizes depending on the
intended application.
Compartmentalization using partial physical
barriers that contain the cells during implantation but
are open to the surrounding cells and tissue components
in one or more places includes, for example, devices
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having shapes and structures in the form of a ring,
cylinder, horseshoe, planer porous structures or any
variation thereof which functionally results in
containment of the cells at implantation. A specific
example of a prosthetic chamber that compartmentalizes
cells using partial physical barriers is a ring of
polytetrafluoroethylene (PTFE). Compartmentalization can
also be accomplished thraugh the use of a three-
dimensional biomatrix containing the cells to be
implanted, beads or particles coated with cells being
attached through receptor ligand interactions as well as
any combination of such compartmentalization devices and
biomatricies. Examples of biomatxix include
extracellular matrix, including for example, collagen
gels. An example of a combination of
compartmentalization de~ri.ses includes cells casted in a
collagen gel contained within a PTFE ring.
Complete encasement of cells includes, for
example, the use prosthetic chambers constructed of
semipermeable materials, such as membranes, that are
barriers to cells but allow passage out of the barrier of
nutrients, cell by-products, growth factors and secreted
gene products. For example, cells can be placed into an
ampule or sack constructed of this material and then
physically sealed. The semipermeable barrier also can be
coated with a biomatrix such as ext.racellular matrix, or
the cells can be contained in a biogel matrix or attached
to particals coated with extracellul.ar matrix components
or cell attachment fragments thereof, for example.
As used herein, the term "effective amount"
when used in reference to the number of cells secreting a
gene product is intended to mean the number of cells that
can be implanted and become engrafted so as to secrete a
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Inhibiting the expression of a target gene in the genome of a plant
may be desired in many purposes. Many transcription factors known by the
man skilled in the art play a role in the control of metabolic pathways
(starch,
lipids, amino acids...) or are involved in the plant development, or in the
plant
s sensibility to pathogen. For example, blocking a gene whose expression is
necessary for pollen or another formation (e.g. the Ms-41-A transcription
factor,
as above described) produces male sterility. Blocking the gene controlled by
the AP-3 transcription factor also leads to male sterility. As another
example,
blocking the gene which codes for the enzyme which catalyses the conversion
~o of sugars to starch can be used to produced sweet corn (see EP 475 584).
It is also possible to obtain transgenic plants with enriched
content in lysine by using, according to the invention, the opaque 2
transcription factor (Schmidt et al., 1990), involved in the control of the
expression of certain zeins. One could further use Myb-related transcription
~s factors involved in the control of anthocyanin biosynthesis in flowers
(Martin et
al., 1991 ; Matin, 1997), to madify their colour. Use of other members of Myb-
related transcription factors playing a role in the regulation of
phenylpropanoid
and lignin biosynthesis (Tamagnone et al., 1998) could also be interesting.
Some others could be involved in cellular development and senescence.
The chimeric construct, fusion of the repressor domain to the
plant transcription factor or part of it, can be advantageously used to
identify
essential protein-protein interaction domains and interacting protein partners
in
planta in transgenic plants.
2s In a preferred embodiment, expression of a chimeric protein
comprising the repressor domain in fusion to a plant transcription factor is
known to cause a dominant phenocopy. By the deletion of increasing parts of
plant sequences encoding the transcription factor and their repression in
fusion
to the repressor domain in transgenic plants, essential protein domains can be
3o identified. These are DNA-binding domains but also others, like protein-
protein
interaction domains, if the transcription factor is part of multi-component
complex. The method according to the invention is therefore also suitable to
study protein-protein interactions in planta.
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implanted in an individual can provide therapeutic
effects in vivo.
The invention provides a method of systemic
delivery of a gene product. The method consists of
implanting within the wall of the digestive tract of an
individual a prosthetic chamber containing an effective
amount of cells secreting said gene product.
The methods of the invention employ cell
engraftment as a vehicle for the systemic delivery of a
gene product. Long-term engraftment ol: cells expressing
a desired gene product is facilitated through the use of
a prosthetic chamber. The chamber :is surgically
implanted at the site of engraftment and cells are seeded
into the chamber either during or previous to
implantation. The function of the prosthetic chamber is
to contain cells at the site of implantation, which
provides an initial favorable microenvironment for cell
viability with an increase the engraftment efficiency of
implanted cells.
The location of the prosthetic chamber can be
essentially any tissue an an individual that is
accessible to surgical procedures and will depend on, for
example, the type of prosthetic chamber used, the cell
type being implanted and the gene product or expression
elements employed for production of the gene product.
Those skilled in the art will know, or can determine a
suitable location for implantation given the teachings
and guidance provided herein. Generally, a suitable
location for implantation of the prosthetic chamber is a
tissue, or region of a tissue which is adeguate:Ly
vascualrized and capable of supplyi:~g nutrients and
growth factors necessary for' cell survival. Such tissues
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can include, for example, muscle including skeletal and
smooth muscle, connective tissue, epithielia and
endothelia cell linings, basement membrane structures,
epidermis and dermis.
Certain tissues and organs have morphologies
that provide additional advantages for the use of
prosthetic chambers as described herein because they
naturally form a stable compartment which further
facilitates the function of the chamber. Implantation
into these regions allows for the use of a broad range of
prosthetic chamber types. 'tissues which exhibit
favorable morphologies include essentially any tissue in
which there is a plane between two :Layers of tissue which
are vascularized and can stably support the surgical
insertion of a prosthetic chamber. For example, the
layers of the digestive tract is one such tissue lacation
that exhibits a morphology favorable for implantation.
Briefly, the wall of the= digestive tract is composed of
four layers. The first, outermost :layer is a thin layer
of mesothelium and connective tissue and is known as the
tunica serosa or seros<~,. The next layer inward is the
tunics muscularis, composed of muscle layers, which is
followed by a second 1<~yer of connective tissue known as
the submucosa. The innermost layer also contains
connective tissue and :is additionally lined with a layer
of epithelial cells. rchis inner layer of the digestive
tract is known as the mucosa. All cyf these layers are
well. vascularized which wi.l.l support. cell growth and
viability. Therefore, a prosthetic chamber can be
implantated into, or be=_tween any of the above .layers and
seeded with cells secreting a desired gene product to
achieve systemic delivery of the product.
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A prosthetic chamber can be implantated into
any of the above described tissues or morphological
tissue layers to form a microcompartment for cells
secreting a desired gene product. Insertion at a plane
between tissue layers :is one location which can be used
for the efficient implantation of a prosthetic chamber.
For example, the plane between any of the four digestive
tract layers described above are easily accessable by
routine surgical procedures, can be separated without
substantial damage to the morphological structure or
biological function of the tissue layer and inherenty
provide a natural crevice once segregated for which a
prosthetic device can tie stably placed and seeded with
cells. For example, the muscle cel'~. bed at the plane
between the tunics muscularis and the tunics serosa on
the stomach can serve as a location to receive a
prosthetic chamber occupied by cells expressing a gene
product of interest. This tissue site is well
vascularized and will therefore provide nutrition for
implanted cells. The long-term survival of implanted
cells requires access to nutrients such as oxygen from
the blood supply. As discussed further below, smooth
muscle cells are a normal part of the architecture of
this site. Modifying this cell type to secrete the
desired gene would therefore provide additional
advantages for the survival of transplanted cells because
they are a normal constituent of the targeted area. A
specific example of impimplanting vascular smooth muscle
cells retrovirally transduced to express erythropoietin
and G-CSF is described further below in the Examples.
Implantation of a prosthetic chamber can be
accomplished by surgical placement cf the chamber into
any of the tissues or morphological tissue layers
described above. Briefly, the chamber is implanted by
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making an incisian at the entry site of the appropriate
depth and length to expose she target tissue and
physically placing the chamber within the tissue
incision. Alternatively, the target tissue can be
externalized, followed by one or more incisions until an
area in the target location has been created for
implantation. Methods for implanting a wide variety of
artificial devices and prothesis as well. as cell, tissue
and organ transplantation and engraftment procedures are
well known to those skilled in the art. Similarly, a
wide variety of surgical methods applicable for the
treatment of diseases, diagnostic indications and
cosmetic procedures are also well known to those skilled
in the art and are all applicable surgical methods which
can be used for implanting a prosthetic chamber of the
invention. For example, the use of endoscopes is well
known to those skilled in the art and affords a minimally
invasive procedure to access and perform surgery on the
stomach and intestin. ~larious modification of and
combinations of such mei~hods known to those skilled in
the art similarly applicable for implanting one or more
prosthetic chambers in an individua.L far the systemic
delivery of a gene product. Specif:LC examples of a
surgical procedure which can be used far implanting a
prosthetic device and seeding with cells modified to
express a desired gene product is described further below
in the Examples.
Therefore, the invention provides a method of
systemic delivery of a gene product where a prosthetic
chamber containing an effective amount of cells secreting
said gene product is implanted within ar proximal to a
connective tissue layer of the digestive tract of an
individual. The location cao be, for example, between
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the tunica serosa and the tunica musclaris of the
digestive tract wall.
The invention also provides a method of
systemic delivery of a gene product by implanting a
5 prosthetic chamber containing an effective amount of
cells secreting said gene product where the prosthetic
chamber has an outer diameter or width that is greater
than about lOmm, preferably between about 7.0-lOmm, more
preferably between about. 3-6mm. The prosthetic chamber
10 can consist of various shapes and can be open at one or
more locations to the surrounding tissue, or it can be
completely enclosed.
Prosthetic chambers used in the methods of the
invention can be of various shapes and sizes so long as
15 they function to restrain cells seeded into the chamber
at the time of implantation and can be surgically
manipulated into a desired location within the recipient
individual. As described previously, the chamber can
either partially or completely encase t:he cells to
provide a beneficial microenvironment for cell growth,
viability and long-term engraftment. Prosthetic chambers
which particially encase seeded cells include chambers
open at one or more locations to the surrounding tissue.
Such chambers exhibit the advantage in that they allow
for expansion of the engrafted ells by proliferation and
migration out of the chamber into the surrounding tissue
regions.
Shapes of such prosthetic chambers include, for
example, a ring, horseshoe and cylinder or any geometric
shape or motif that contains an inner region which can
contain seeded cells and an outer barrier which serves to
restrain the cells. For example, a ring having an inner
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diameter of about 4mm for seeding cells and an outer
diameter of about 6mm is one example of a prosthetic
chamber that is partially open to surrounding tissue and
capable of containing cells at the site of implantation.
Cells seeded into the center of the ring are initially
restrained by the outer barrier at implantation. As the
implanted cells engraft and expand they are able to
migrate out of the ring and over the outer barrier
forming a patch of stably engrafted cells. Other shapes
having similar functional features such as those listed
above as well as squares, hexagons, rectangles, triangles
and the like also can be used for a prosthetic chamber in
the methods of the invention. Therefore, the shape of
the chamber is unimportant so long as the chamber
25 functions to contain cells seeded at the site of
implantation.
Prosthetic chambers having various alternative
forms to those described above also can be used in the
methods of the invention. For example, prosthetic
2o chambers composed of a planar, porous material will
similarly function to contain cells at the site of
implantation, allow access to the surrounding tissue and
exchange of nutrients, factors and cell by-products.
Cells can be seeded into the pores which completely, or
25 partially extend, through the planar material. One
advantage with this structure for a prosthetic chamber is
that the surface for cell containmE~nt is increased
portional to the number and pores. Additionally, cells
can be seeded onto the outer so rfa<:es of the plane by,
3o for example, coating it with cell adhesion proteins yr
substrates and allowing the cells to ;attach through
receptor-ligand interactions. Concave disks of various
shapes and depths can similarly furocr.ion as a prosthetic
chamber to contain cells at the sit::e of implantation. As
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with porous chambers, concave disks can be coated with
adherent proteins to facilitate cell attachment at the
time of implantation or, alternatively, preseeded with
cells prior to implantation.
Prosthetic chambers that can be used in the
methods of the invention also can be of various three-
dimensional shapes and sizes. For example, the chambers
can be cylinders and cones which have an inner area for
containment of the cells and an outer barrier.
Additionally, chambers can be particles or spheres which
can be coated which adherent cells ;prior to or during
implantation. The particles or spheres can be porous or
textured so as to increase the surface area and therefore
the number of cells r_apable of being implanted using such
a chamber. Therefore, the chambers which can be used in
the methods of the invention are not limited to a
particular geometric shape, structure or three-
dimensional form.
The selection of a particular shape or form for
use in the methods of the invention will depend, for
example, on the site of implantation, number cells to be
seeded and surgical preference by those skilled in the
art. For example, if the prosthetic chamber is to be
implanted between tissue layers, such as between the
tunica serosa and the tunica muscularis of the digestive
tract, those skilled in the art can select, for example,
a ring, horseshoe, other geometric shape, or other planar
chamber that can be inserted between the tissue layers
with minimal damage and preturbat.i.on of the surrounding
tissue. Those skilled in the art. will know, or can
determine, what shape and structure of the prosthetic
chamber is appropriate fear a particular tissue location.
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Prosthetic ctuambers which completely encase
seeded cells include chambers such as spheres, ampules,
and sacks constructed ~:of semipermiable barriers. Such
chambers can be advant~:~geous when using non-autologous
cells because they physically block host immune cell
attack and therefore reduce graft rejection by this
mechanism. The use of such completely enclosed
prosthetic chambers therefore allows for the implantation
of non-autologous cells and reduces or eliminates the
need for immunosuppressa.ve drugs which compromise the
recipient's immune sysr_em. The shape of such chambers
can additionally be any of those described previously
except that the exposec:~ areas of the previously described
chamber shapes would instead be enclosed with a
semipermeable barrier. for example, a ring can be used
where the top and bottom planes are enclosed with, for
example, a semipermeab:Le membrane, thus forming an
enclosed cylinder or oval-shaped structure having cells
seeded into the internal area prior to implantation.
The prosthetic: chambers used in the methods of
the invention can be of various sizes and will depend on
the desired number of :ells to be implanted, in addition
to the site of implant<~tion and associated considerations
described above in reg<srd to selection of the shape of
the chamber. For example, a ring having an inner
diameter of about 9mm will accommodate about 1x:.0 or
greater number of cells. Larger chambers larger will
accommodate proportionally more cells and conversely,
smaller chambers can bs~ used to implant proportionally
less cells. In additi<:~n, the total number o:f cells car.
be substantially increased by about 2- to 10-fold, or
more if porous or text'.ired chambers are used. The
chambers therefore can k}e essentially any size which the
tissue at the site of implantation c~an physically
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accommodate and which is in acceptable medical parameters
for implantation of a prosthetic device.
For example, a prosthetic chamber between about
7 and lOmm or greater in outer diameter or width and as
small as 3 to 6mm can be accomodiated at the tissue plane
between the tunica serosa and the tunica muscularis. The
inner diameter or width of a prosthetic: chamber used in
the methods of the invention similarly can be within
these same size ranges and can depend, for example, an
the type of material used and the methods of fabrication.
The height of the prosthetic chamber is generally about
3mm to allow sufficient diffusion of oxygen into the
implanted cell area. However, the height of a chamber
can be as small as 1-2mm and as large as 9-5mm or
greater. Moreover, the height can be of various other
sizes depending on the site of implantation and the shape
and surface area exposed to the surrounding tissue and
vasulature. Those skilled in the art will know how to
vary the height of the prosthetic chamber to achieve
sufficient diffusion of axygen and other nutrients to
maintain viability and support engraftment of the
implanted cells given the teachings and guidance provided
herein.
The above described prosthetic chamber sizes
will accommodate populations of cells from about 1x10' or
smaller. The size of the cell population to be implanted
can be, for example, as small as 1x10' or less. However,
larger populations such as 1x10', 1x10, 1x10' and ,yxl0~
are, in general, the amount of implanted cells that will
secrete a sufficient amount of the desired gene product
to accomplish the targeted functional effect. Therefore,
any of the prosthetic chambers described above, as well
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zo
as others known to those skilled in the art. are
applicable for use in the methods of. the invention
The number of cells required for an effective
amount will vary depending on the gene product being
secreted, its expression and secretion level into the
extracellular space arid its inherent biochemical
activity. For example, a gene product that has a high
affinity for substrate or ligand will inherently require
less amounts to be secreted to achieve the same level of
l0 activity when compared to a lower affinity homologue.
Similarly, a gene product with a longer half life also
will require less amounts or rate of secretion to achieve
comparable activities or accumulation levels to a shorter
lived gene product. Therefore, an effective amount of
cells will vary depending on inherent properties of the
gene product to be secreted and expression
characteristics of the modified cells, all of which are
criteria well know to those skilled in the art.
Therefore, the invention also provides a method
of delivering a gene product systemically by implanting
an effective amount of cells secreting the gene product
where the number of cells is a population of about 1x20'
cells or greater, preferably about 1x10' cells or greater,
more preferably about 1x10' cells or greater.
Fabrication materials for the prosthetic
chambers can be of any solid, porous or degradable
material so long as it is biocompatable. For example,
the chamber composition can include surgical grade
metals, synthetic polymers and b~~.opolymers. Examples of
synthetic fabrication material is polytetrafluoroethylene
(PTFE), gortexG~ and carbon fibe r Surgical grade metals
include, for example, titanium and stainless steel
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21
whereas bivpolymers can included, for example, collagen,
fibronectin, proteoglycans and active fragments thereof.
The prosthetic chambers can additionally include a wide
variety of intracellular and systemic biomolecules that
augment cell viability and proliferation of implanted
cells. Such biomolecules can include, for example,
growth factors and hormones. Additionally, prosthetic
chambers containing semi-permeable barriers can be
composed of, for example, natural or synthetic membranes
with a pore size that excludes cell-cell contact.
Generally, a pore size of about 0.22 mm is sufficient to
allow exchange of macromolecules such as polypeptides and
proteins. However, other pore sizes can also be used
without affecting the function of the chamber ar of the
implanted cells. Specific examples of chambers made from
some of these materials are described below and in the
Examples. Other materials known to those skilled in the
art also can be used for the prosthetic chambers used in
the methods of the invention.
Briefly, prosthetic chambers used in the
methods of the invention can be fabricated with PTFE.
This material is non-immunogenic, easily fabricated into
various shapes, sizes and formats, can be prepared for
implantation using methods well known to those skilled in
the art for sterilization arid handling and can be
manipulated by routinely surgical procedures for
implantation. Prosthetic chambers also can be fabricated
using various lengths of 18g cerclage wire, or surgical
grade stainless steel wire, which is bent into a "U" or
horseshoe shape to restrain cells seeded into the
chamber. Modification to the prosthetic chambers can
additionally be made t:o help maintain the shape and
volume of the chamber. For example, short lengths or
disks of about 0.5-lcm in length or diameter and about
CA 02330172 2001-11-30
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22
lmm thick PTFE or other compatible material can be
inserted in the chamber after the cells are introduced to
achieve this effect. A fur~.her modification includes
casting the cells that are to be grafted into the chamber
in a contracting collagen matrix which serves to both
concentrate the cells and to hold the cells in place
after engraftment. This type of prosthetic chamber is
applicable with any bi.ocompatible material that can be
manipulated into a shape to contain cells. The inner
diameters of any of such chambers can exceed 10 mm and
allows for the engraftment of at least about 1x10' or more
modified cells.
Systemic delivery of a desired gene product
utilizes cells as a vehicle for delivery and long-term
production of the gene product in the recipient
individual. The cells can be modified ex vivo to express
in a secreted form the desired gene product, or
alternatively, the imply nted cells can be natural
producers of the gene product. Cell. implantation and
engraftment is facilitated using the prosthetic chambers
as described above. The cell type t:o he implanted can be
any cell type, population of cell types and heterogeneous
mixtures, that ar.e compatible with the site of
implantation and the recipient's major. histocompat:ibility
(MHC) antigens responsible for graft rejection.
Compatibility includes modification of the implanted
cells or treatment of the recipient so as to allow self-
recognition by the recipient's immune system or to down
regulate the recipient'; immune system by
immunosuppressive agents. Therefore, and as described
further below, selection of the cell type to be implanted
follows criteria for ce.l.l., tissue and organ engraf~ment
and/or transplantation which is well known to those
skilled in the art.
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Cell types to be selected for generating the
modified cells used in the methods of the invention are
those which are capable of polypeptide synthesis and
secretion and compatible with the recipient's immune
system and the site of implantation. With the exception
of highly specialized cell types, such as red blood
cells, the large majority of cells meet these criteria
and can be used for constructing the modified cells used
in the methods of invention. Alternatively, cells which
naturally express the desired gene product can be
obtained, for example, from a donor individual or ;.issue
and implanted directly without the need for genetic
modification.
The cell type chosen for .modification is
selected according to the biological characteristics of
the cell and according to gene expression criteria well
known in the art. For example, abjective criteria such
as the ease of culture efficiency, the ease of genetic
modification and other routine cellular and molecular
manipulations can be used to evaluate and select the cell
type for modification. Those cell types which can be
passaged in culture for multiple generations without
substantial loss in viability are preferable candidates
for modification as systemic delivery vehicles for a
desired gene product. As will be described further
below, such cell types include, for example, both primary
cells as well as cell lines. Additionally, criteria such
as the proliferation characteristics can also be
evaluated for selection of the cell type to be modified.
Cell types are additionally selected according
the efficiency with which they can be modified to express
and secrete a gene product in the methods of the
invention. Cell types that can be readily modified and
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24
selected for the expression of the introduced gene or
genes by any of a variety of methods known in the art are
applicable for constructing the implantable cells of the
invention. Availability of promoter and regulatory
elements can also be included as a criteria for selecting
a particular cell type for modification. Such
characteristics and criteria are routine and well know to
those skilled in the a.r1-_.
Various combinations of the above exemplary
characteristics as well .~s other characteristics can
additionally be used far selecting ,~ cell type to modify.
For example, if the objective is to achieve a particular
level of gene praduct, sc~c:retion using a relatively small
number of cells, then a cell type that can be efficiently
modified to yield high levels of expression can be
selected to achieve the desired result. In contrast, if
cell number is nat a limiting factor, then it can be
desirable to base selection of the cell type on favorable
growth or proliferation characteristics. Additionally,
various expression elements can be utilized to augment or
modulate the level of expression. and secretion so as to
complement advantageous characteristics or to overcome
any deficiencies of the chosen cell type for
modification. Such criteria and characteristics are well
known or can be determined by those skilled in the art.
The cells selected for use in the methods of
the invention can therefore originate from essentially
any tissue or organ. However, the engraftment efficiency
can be inherently augmented by selecting a cell type or
composition of cell types that are natural constituants
of the tissue where the prosthetic chamber is to be
implanted. For example, cellular constituents of the
layers of the digestive tract Include connective tissue
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of mesothelial origin, muscle and epithelial cells.
Therefore, selecting one of these cell types for
implantation at the corresponding tissue layer will yield
engraftment into an environment of similar or identical
5 cells and provide a compatible habitat for cell. viability
and growth. A specific example, is the implantation of
modified smooth muscle cells between the layers of the
tunica serosa and the tunica muscularis of the digestive
tract because the latter. layer is composed primarily of
10 smooth muscle cells.
Another specific example of a cell type that is
useful for engraftment with the methods and prosthetic
chambers of the invention is a fibroblast cell.
Fibroblast cells can be obtained from a variety of tissue
15 sources including, for example, skin, liver, muscle or
arterial tissue. Fibroblast cells are also advantageous
in that they are easily obtained and isolated, routinely
modified, and can proliferate to higher densities within
a graft. For example, a 10 cm= area within a prosthetic
20 chamber can contain 10' fibroblast cells, or up to 20
layers of cells, and can secrete essentially any desired
gene product at effective amounts to achieve
therapeutically beneficial results.
Therefore, the invention provides a method of
25 systemic delivery of a gene product where cells secreting
the gene product are smooth muscle cells or fi.broblasts.
The gene product can be constitutively secreted, or
alternatively, it can be secteted by i.nducible
regulation.
For implanting primary cells, a tissue and
therefore a site for implanting a prosthetic chamber,
should be selected that is easily accessible arzd contains
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26
cells that exhibit desirable growth and expression
characteristics such as those described herein.
Additional considerations when selecting a tissue source
include choice of a tissue that contains cells that can
be isolated, cultured and modified to secrete a desired
gene product. In addition to the cellular constituents
of the digestive tract layers, other examples of cell
types that can be modified t.o secrete a desired gene
product include, for example, muscle (smooth, skeletal or
cardiac), fibroblast, liver, fat, hematopoietic,
epithelial, endothelial, endocrine, exocrine, kidney,
bladder, spleen, stem and germ cells. Other cell types
are similarly known in the art that are capable of being
modified to secrete gene products and can similarly be
obtained or isolated from a tissue source from a
recipient or donor individual. Although human tissue
sources are advantageous for therapeutic purposes, the
species of origin of the cells can be derived from
essentially any mammal, so long as the cells exhibit the
characteristics that a7.low for secretion of the gene
product of interest.
Cells to be used in a prosthetic chamber car. be
adherent cell types, which can require isolation methods
that temporarily digest or release the cells from their
surrounding tissue. for example, smooth muscle cells can
be obtained from a segment of veno~.~s or arterial tissue.
The smooth muscle cells can be obtained following
enzymatic digestion in trypsin and co:llagenase and
purified by positive selection using muscle cell-specific
antigens such as von Willebrand factor (hejnieks et al.,
Bloc,_d, 92:1-7 (1998). Alternative7.y, cells can be
isolated following digestion and further purified by
centrifugation. The centrifugation can be performed in
the presence of a gradient such as a sucrose gradient,
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27
which would allow for further separation of cell
populations based on their density, Methods for the
isolation of primary cells from a tissue source are well
known in the art (see, for example, Freshney, Animal Cell
~~ltm re: A Practical Ap~oach, 2nd ed., IRL Press at
Oxford University Press, New York (1992). Maintenance of
the cells prior to modification and implantation can be
as a cell suspension, adherent cell culture or as organ
culture. Conditions for the maintenance and culture of
l0 primary and clonal cells are well known in the art.
For therapeutic applications, the above cell
types are additionally chosen to be implantable in an
individual and remain viable in visrr~ without being
substantially rejected by the host immune system.
15 Therefore, the donor origin of the cell type is a further
characteristic that should be evaluated when selecting
cells for therapeutic application. Those skilled in the
art know what characteristics should be exhibited by
cells to remain viable following implantation. Moreover,
20 methods well known in the art are available to augment
the viability of cells following implantation into a
recipient individual.
One characteristic for an implanted donor cell
type is to exhibit substantial immunological
25 compatibility with the recipient individual. A cell is
immunologically compatible if it is either
histocompatible with recipient host antigens or if it
exhibits sufficient similarity in cell surface antigens
so as not to elicit an effective toast anti-graft immune
30 response. Specific examples of immunologically
compatible cells include autologous cells isolated from
the recipient individual and allogene:ic cells which have
substantially matched major histocompatibility (MHC) or
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28
transplantation antigens with the recipient individual.
Immunological cornpatibility can be determined by antigen
typing using methods well known in the art. Using such
methods those skilled in the art will know or can
determine what level of antigen similarity is necessary
for a cell or cell population to be immunologically
compatible with a recipient individual. Tolerable
differences between a donor cell and a recipient can vary
with different tissues and can be readily determined by
those skilled in the art.
In addition to selecting cells which exhibit
characteristics that maintain viability following
implantation into a recipient individual, methods well
known in the art can be used to reduce the severity of an
anti-graft immune response. Such methods can be used to
further increase the in vivo viability of immunologically
compatible cells or to allow the in v~.vo viability of
less than perfectly matched cells or of non-
immunologically compatible cells. Therefore, for
therapeutic applications, it is rlOt necessary to select a
cell type from the recipient individual to achieve
viability of the modified cell following implantation.
Instead, and as described further below, alternative
methods can be employed which can be used in conjunction
with essentially any donor cell t.o confer sufficient
viability of the modified cells to achieve a particular
therapeutic effect.
For example, in the case of partially matched
or non-matched cells, immunosuppressive agents can be
used to render the host immune system tolerable to
engraftment of the implanted cells. The regiment and
type of immunosuppressive agent to be administered will
depend on the degree of MHC similarity between the
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29
modified donor cell and the recipient. Those skilled in
the art know, or can determine, what level of
histocompatibility between donor and recipient antigens
is applicable for use with one or more immunosuppressive
agents. Following standard clinical protocols,
administration and dosing of such immunosuppressive
agents can be adjusted to improve efficiency of
engraftment and the viability of the cells of the
invention. Specific examples of immunosuppressive agents
useful for reducing a host anti-graft immune response
include, for example. cyclosporin, corticosteroids, and
the immunosuppressive antibody known in the art as OKT3.
Another method which can be used to confer
sufficient viability on partially-matched or non-matched
cells is through the masking of the cells or of one or
more MHC antigens) to protect the cells from host immune
surveillance. Such methods allow the use of nan-
autologous cells in an individual. Methods for masking
cells or MHC molecules are well known in the art and
include, for example, physically protecting or concealing
the cells, as we:l.l as disguising them, from host immune
surveillance. Physically protecting t_he cells can be
achieved, for example, by encapsulating the cells within
a semi-permeable prosthetic chamber as. described
previously. Such a barrier prevents contact of host
immune cells such as T-cells with the cells contained
within the chamber but. still allows secretion of the
desired gene product into the ext.race:Llular space and
circulation system. Alternatively, antigens can be
disguised by treating them with binding molecules such as
antibodies that mask surface antigens and prevent
recognition by the immune system.
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Immunologica:lly naive cells also can be used
for constructing the impl.antable cells of the invention.
Immunologically naive cells are devoid of MHC antigens
that are recognized by a host anti-graft immune response.
5 Alternatively, such cells can contain one or more
antigens in a non-recognizable form or can contain
modified antigens that faithfully mirror a broad spectrum
of MHC antigens and are therefore recognized as
self-antigens by most MHC molecules. The use of
10 immunologically naive cells therefore has the added
advantage of circumventing the use of the above-described
immunosuppressive methods for augmenting or conferring
immunocompatibility onto partially or non-matched cells.
As with autologous or allogeneic cells, such
15 immunosuppressive methods can nevertheless be used in
conjunction with immunologically naive cells to
facilitate viability of the glucose-regulated insulin
producing cells.
An immunologically naive cell, or broad
20 spectrum donor cell, can be obtained from a variety of
undifferentiated tissue sources, as well as from
immunologically privileged tissues. Undifferentiated
tissue sources include:, for example, cells obtained from
embryonic and fetal tissues. An additional source of
25 immunologically naive cells include stem cells and
lineage-specific progenitor cells. These cells are
capable of further differentiation to give i:ise to
multiple different cell types. Stem cells can be
obtained from embryonic, fetal and adult tissues using
30 methods well known to those skilled i.n the art. Such
cells can be used directly or modified further to enhance
their donor spectrum of activity.
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31
Immunologically privileged tissue sources
include those tissues which express, for example,
alternative MHC antigens or immunosuppressive molecules.
A specific example of alternative MHC antigens are those
expressed by placental cells which prevent maternal
anti-fetal immune responses. Additionally, placental
cells are also known to express local immuno-suppressive
molecules which inhibit the activity of maternal immune
cells.
An immunologically naive cell or other donor
cell can be modified to express genes encoding, for
example, alternative MHC or immuno-suppressive molecules
which confer immune evasive characteristics. Such a
broad spectrum donor cell, or similarly, any of the donor
cells described previously, can be tested for
immunological compatibility by determining its
immunogenicity in the presence of recipient immune cells.
Methods for determining immunogenicity and criteria for
compatibility are well known in the ax't and include, for
example, a mixed lymphocyte reaction, a chromium release
assay or a natural killer cell assay. Immunogenicity can
be assessed by culturing donor cells together with
lympohocyte effector cells obtained from a recipient
individual and measuring the survival of the donor cell
targets. The extent of survival of the donor cells is
indicative of, and correlates with, the viability of the
cells following implantation.
Once a cell type has been selected as described
above, cells expressing a desired gene product in a
secretable form are generated by introducing a vector
expressing the encoding gene into the selected cell type.
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An expressible nucleic acid encoding a desired
gene product can be constructed using methods well known
in the art. Such methods are described in, for example,
Sambrook et al . (Molecul ar Cl,~n; ng: ~~~~a~t ,~,y Mai La1 ,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor
(1989)) and Ausubel et al. (~~;~rent Protocols in
Molecular Bioloav, John Wiley & Sons, LVew York (1998)).
For example, a nucleic acid sequence encoding the gene
product can be obtained using polymerase chain reaction
(PCR) to amplify the sequence from an appropriate tissue
or cell type. Encoding nucleic acids can be obtained by
other methods known to those skilled in the art as well
as by cloning or chemical synthesis using sequences
available in scientific databases.
Once obtained the encoding gene is
operationally linked t:o express.ion and regulatory
elements which direct expression and ;>ecretion of the
gene product into the extracellu.lar space. Such promoter
and regulatory elements are also well know in the art and
are selected for compatibility with the cell type to be
implanted. For example, retrovir.al. promoter and
enhancers achieve constitutively high levels of
expression in a wide variety of different cell types,
including smooth muscJ.sy cells. GJhereas a constitutive
promoter yielding relatively strong expression levels in
fibroblasts can be selected from a variety of extracellar
matrix proteins such as fibronectin, collage and laminin,
from structural genes such as actin, tubulin and
intermediate filaments and from general housekeeping
genes such as those in the glycolysis pathway. Selection
of compatible express.i.<:>n and regulatory elements for a
particular cell type a~.so is wel:L known to those skilled
in the art.
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Promoters can be constitutive such as those
described above, or alternatively they can be inducible.
A specific examples of :inducible promoters is the glucose
regulated promoter describe below and in the Examples.
For example, glucose-regulated expression of a gene
product of interest can be achieved using a variety of
promoter elements that control expression of a downstream
gene in response to changes in levels of glucose.
Elements that are induc.ible by glucose generally exhibit
low activity in the absence of glucose and are up-
regulated in the presence of. increased glucose or a
metabolite within a glu~.:ose-associated pathway. For
example, it is known that TGF-a promoter activity is
responsive to glucose (~lcClain et al., Proc. Natl. Acad.
~,ci. USA 89:8150-8154 (1992): and Raja et al., Mol.
E~~dc~crinol. 5:514-420 (1991)). Also, sequences from
other glucose-responsive promoter elements can be used
such as promoter elements from wild-type insulin,
fibroblast growth factor (FGF), epidermal growth factor
(EGE), PC2 or PC3 genes (Sander et al., Proc. Natl. Acad_
Sci. USA 95:11572-11577 (1998)). Other glucose
responsive promoter elements can be found, for example,
in genes for acetyl-CoA carboxylase, 6-phosphofructo-2-
kinase, and L-type pyruvate kinase (Zhang and Kim, Arch.
,~?ochem. Bionhvs. 15:227-232 (1997): Dupriez and
Rousseau, D~,A Cell Ri_o1-. 16:1075-1085 (1997); Antoine et
al., ~. Bioj. Chem. 272:17937-17943 (1997); and Kennedy
et al . , ~,~o,l~ Chem.. 272 : 20636-2069U ( 1997 ) ) . A wide
variety of other inducible promoters applicable for use
in the methods of the invention are well known in the
art. Selection of a particular inducible promoter will
depend, for example, on the desired level of expression
and as well as the choice o.f inducer to administer to a
recipient individual.
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39
Similarly, selection and operational linkage of
the encoding nucleic acid to regulatory sequences for
processing and secretian into the ext:racellular space is
similarly well known in the art. E'or delivery of one or
more desired gene products to the circulatory system it
is necessary for the product to be secreted. Therefore,
the encoding nucleic acids should contain a signal.
sequence for routing to the endoplasmic reticulum and
secretion outside of the cell. The signal sequence can
to be derived from the authentic gene for the product of
interest, or alternatively from a heterologous gene and
linked in frame for secretion into the extraceilular
space. Signal sequences and methods of using them are
well known in the art.
Vectors used in the methods of the invention
can be any vector computable with the host cell or tissue
where the desired gene product is to be expressed. For
example, the vectors can be plasmi.d or viral based
vectors. The vectors can be known, or constructed from
components well known :in the art. Tn the case of a viral
vector, the vector can contain, for example, at least one
viral long terminal repeat and a promoter sequence
upstream and operably linked to a nucleotide sequence
encoding the gene product of interest, followed by at
Z5 least one viral long terminal repeat and polyadenylation
signal downstream of the sequence encoding the gene
product of interest. Where multiple gene products are to
be expressed, a single vector can be used which encodes
more than one gene of interest, ear_h gene of interest can
be expressed as separate transcription units.
Alternatively, multiple genes can be transcribed as a
single transcription unit containing internal ribosome
entry sites that allow expression of the genes from a
polycistronic messenger RNA (Adam et al., J. Virol.
CA 02330172 2001-11-30
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65:4985-4990 (1991)). Additional. nucleic acid sequences
can be inserted into the vector using methods well known
in the art.
Representative retroviral vectors suitable for
5 use in the invention and methods for their design are
described for example, in Osborne and Miller, Proc. Natl.
8cad Sci. USA. 85:68'il-6855 (1988); Osborne et al.,
c. Natl. Acad. Sci. USA. 92:8055-8058 (1995); Ramesh
et al., Nun. Acids. Rp,~~., ?.9:2697-a?700 (1996): and Hock
10 et al., lood. 74:876-881 (19891. Other vectors may also
be used and are well known in the art, such as lentiviral
vectors, DNA vectors, adenoviral vectors, pseudotype
retroviral vectors, Epstein-Barr viral vectors, adeno-
associated viral (AAV', vectors, vesicular stomatitis
15 virus-g (VSV-g), VL30 vectors, and liposome mediated
vectors. AAV vectors provide an advantage in that they
integrate into the host cell chromosome and do not
substantially cause an immune response from the recipient
host individual. Representative pl.asmid vectors include,
20 pcDNA and various other mammalian cwell expression vectors
which are commercially available through a variety of
sources.
Methods for introducing such vectors into a
cell are also well known in the art. (see for example,
25 Osborne et al., supra (1995)). One method of introducing
a vector into a cell :is by transfec:tion of plasmid or DNA
vectors. Transfection methods are well known in the art
and include, for example, calcium phosphate
precipitation, electroporation, liposome-mediated
30 transfection, and microinjection as described, for
example, in Sambrook .at al. supra (1989) and Ausubel et
al., supra (1998). Alternatively, a retroviral or an AAV
vector can be transduced into a cell. Methods for
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36
transduction of retroviral and adenoviral-type vectors
are also well known in the art and are described further
below in the Examples.
Following transfection or transduction of cells
S with vectors of the invention, the cells are selected
using a selectable marker that is either on the same
vector as the gene of interest or is co-transfected on a
separate vector. Methods of selecting cells for
expression of a selectable marker' encoded by a
transfected vector are well known to those skilled in the
art (see, for example, Ausubel et al. supra (1998)).
Following selection, an isolated population of cells
expressing the desired gene product or products is
obtained.
Verification that the population of cells
expresses and secretes the desired gene product or
products can be determined using methods well known in
the art. For example, a modified population of cells can
be verified for the ability to secrete the gene product
assaying the amount of product secreted into the culture
media under expression-promoting conditions. The can be
measured by, for example, enzyme-linked immunoaffinity
assay, radioimmunoassay or by a functional assay for one
of the known biological functions of t:he gene product.
Such assays are well known to those skilled in the art.
Additional methods of selecting cells expressing the
desired gene product include Northern analysis and
solution hybridization of mRNA obtained from the cells,
in situ hybridization, immunohistology, and
immunofluorescence using antibodies specific for the gene
product. Further selection of a population of cells
suitable for use in the invention can be performed using
in vivo models known to those skilled in the art. Once a
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37
population of modified cells has been obtained, the cells
can be seeded into a prosthetic chamber and implanted
into a recipient individual.
The invention provides a method of systemic
delivery of a gene product wherein the' gene praduct is
selected from the group consisting of c°ytokines, hormones
and growth factors. The secreted gene product can be Epo
and insulin.
The methods of the invention are effective for
expressing essentially any gene product which can have an
effect on a target disease or pathological condition if
it can, for example, effect a change in the phenotypic or
biochemical characteristics of the target tissue's
cellular constituents by direct contact of the secreted
gene product with the target cells, or alternatively, by
indirect contact with one or mare factors, including
other gene products, induced by the secreted gene product
with the target cells. Indirect effects of the secreted
gene product also include, for example, negatively
regulating a factor which plays a detrimental role in the
target disease or pathological condition by inducing down
regulation of that factor through secretion of a negative
regulator using the methods of the invention. Therefore,
the methods for treating a pathology mediated by a
deficiency of a gene product of the invention are
directed to those pathologies having deficiencies in the
expression or regulation of a extracel:lular gene product,
including gene products found systemically, or having
their effect via the systemic system. Those skilled in
the art will know which particular gene product wi.Ll have
therapeutic or beneficia.L effects on a particular disease
and are applicable for use in the methods of the
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38
invention given the teachings and guidance provided
herein.
For example, the methods of the invention can
be used to implant an effective amount of cells secreting
a wide variety of genEy products which reduce the severity
of a number of diseases or pathological conditions.
Exemplarily gene products include, for example, hormones,
cytokines, growth factors, blood clotting factors and
enzymes. A specific example of a clotting factor is
factor IX which can bE:~ used for reducing the severity of
individuals with hemo~>hilia. Glucocerebrosidase is a
specific example of aru enzyme which can be secreted from
modified cells and implanted to treat the severity of
Gauchers disease. Other gene products and categories of
gene products which can be systemically delivered using
the modified cells as implantab.Lc vehic7_es are known to
those skilled in the drt and similarly applicable for use
in the methods so the invention.
Hormones, cytokines and growth factors are
three classes of factors that carr be secreted by
implanted cells into a recipient to achieve a wide range
of beneficial effects, and thus, reduce the severity of
the condition. For example, Epo is a hormone which
stimulates red blood cell p.roduct.ion. Secretion of this
cytokine from engrafted cells following implantation can
be used in conjunctiorn with a variety of diseases and
pathological conditions to augment production of red
cells. Such diseases and pathological conditions
include, for example, organ and tissuEe transplants,
anemia, blood transfusion, renal failure and human
immunodeficiency viru:~ infection.
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Another hormone that can be secreted from
implanted cells to trE:~at or reduce the severity of a
disease is insulin. ':n diabetic individuals blood
glucose can be high due to the lack of insulin
production. Secretiorn of this hormone from engrafted
cells following implantation will facilitate cellular
uptake of blood glucose following digestion of a food in
a diabetic individual. Moreover, the insulin encoding
gene can be placed under the control of a glucose
regulated promoter to achieve regulation of insulin
production that parallels increases in blood sugar and
achieves glucose homes>stasis similar t=o that of normal
individuals. Therefore, the long term engraftment of
insulin producing cells will alleviate the need for
intravenous injection of insulin.
Interleukins are a class of immune-related
cytokines which can be used to treat a wide variety of
diseases and pathological conditions. These molecules
function, for example, in immune cell regulation and can
be used to augment immune responses for the treatment of
immune disorders, including autoimrnune diseases, as well
as for the treatment c>t cancer. Far example, in immune
related disorders, regulatory T cells can be stimulated
with interleukins to augment their ability to induce or
inhibit an immune response. Similarly, in cancer
treatment, immune celi.s can be stinnulated with
interleukins to facilitate immune responses against the
pathological cells or to increase the effectiveness of
other therapies.
Other cytokines which function similarly can
also be used either by themselves ar in conjunction with
an interleukin. ThosE:~ skilled in the art know the
physiological and regi.ilatory effects of a cytokine and
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can therefore select the appropriate molecule or
combination of molecules to effectively treat or reduce
the severity of an immune disorder or cancer. Therefore,
interleukins are described herein as exemplarily
5 cytokines for the treatment of these pathological
conditions and can readily be substituted or combined
with another cytokines known to those skilled in the art
to have an effect on a target disease such as an immune-
related disease or cancer. Other diseases and
10 pathological conditions exist which can similarly be
treated through the implantation and engraftment of cells
expressing a number of different cytokines. The
treatment of these conditions by expressing cytokines
having known therapeutic effects can similarly be
15 accomplished using the method of the invention.
Growth factars such as granulocyte-colony
stimulating factor (G-CSF), granulocyte, macrophage-
colony stimulating factor IGM-CSF), and related factors
which stimulate cell proliferation and differentiation
20 within the hematopoietic cell lineages also can be used
for treatment or reduction in severity of a variety of
diseases and pathological conditions using the methods
described herein. For example, the stimulation of
leukocyte production following bone marrow
25 transplantations can be increased by implanting into the
bone marrow recipient cells expressing G-CSF or GM'CSF.
These cells are necessary for immune surveillance and are
critical for protection against pathogenic organisms and
therefore early survival of a recipient individual
30 following bone marrow ablation and transplantation.
Other factors within the hematopoietic lineage similarly
induce the differentiation and proliferation of other
cell lineages and also can be secreted by implanted cells
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as described herein to stimulate some or all of the cell
lineages of this system.
A variety of growth factors other than the
specific examples listed above also can be used in the
methods of the invention. Such growth factors include,
for example, insulin-like growth factor (IGF), nerve
growth factor (NGF), epidermal growth factor (EGF),
inhibins, tumor growth :factor (TGF), tumor necrosis
factor (TNF), hepatocyte growth factor (HGF), platelet-
derived growth factor (PDGF), fibroblast growth factor
(FGF), stem cell factor, leukemia inhibitory factor and
thrombopoietin. Such factors and their biological
effects as well as those of the intcrleukin family of
cytokines can be found described in" for example,
Molecular Bioloqy and BiotechnoloUV, Ed lt.A. Myers, VCH
Publishers, Inc., New 'York, N.Y., 1995, which is
incorporated herein by reference. Other growth factors,
hormones and cytokines known in the art; can similarly be
used in the methods of the invention to treat or reduce
the severity of a disease or pathological condition. The
selection of a particu.iar factor or factors to be
secreted using the cell implantation methods of the
invention can be made c:liven the tea<~hings and guidance
provided herein together with that which is known in the
art for the treatment of a particular disease.
It is understood that modifications which do
not substantially affec t the activity of the various
embodiments of this invention are also included within
the definition of the invention pro~.rided herein.
Accordingly, the following examples are intended to
illustrate but not limit. the present: invention.
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EXAMPLE I
Long-Term In Yivo Expression of Therapeutic
Levels of Ervthropoietin
This Example describes the use of a prosthetic
chamber for systemic, long-term delivery of Epo from
implanted cells to achieve elevated levels of red blood
cell production.
Smooth muscle cells were used as the
implantation and delivery vehicle for Epo secretion. The
site of implantation was in the digestive tract between
the tunics serosa and the tunics muscularis. Briefly,
expression and secretion of Epo was accomplished from
smooth muscle cells transduced with a retroviral vector
expressing the rat Epo cDNA. The retr.oviral vector,
LrEpSN, was made by inserting an EcoRl:-BamHI fragment of
the rat Epo cDNA into vector LXSN (Usborne et al., 1995,
supra; Miller et al., Biotechniques 7:980 (1989)) A
plasmid containing the rat erythropoietin gene was
obtained from Drs. JPR. Boissel and HF Bunn, and is
describe in Wen et a:L., Blood 82:1507 (1993). The
control vector, LASH, was made by inserting human ADA
cDNA into LPNSN-2, a vector encoding human purine
nucleoside phosphorylase, as described by Hock et al.,
Blood 74:876-881 (1989). Similarly, the amphot.ropic
retroviruses were generated as described by Hock et al.
Rat smooth muscle cells were used for
implantation following transduction with the above
described vectors. Briefly, rat smooth muscle cell
cultures were prepared ;by enzymatic digestion of the
aorta from a male Fisher 394 rat, and the cells were
characterized by positive staining for muscle cell~-
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93
specific actins with HHF35 antibody and negative staining
for von Willebrand factor, which is arr endothelial cell
specific marker (see f:or example, teary et al., Hum. Gene
Ther. 5:1213 (1994)). Primary cultures of rat smooth
muscle cells as well as ecotropic FESUl and amphotropic
PA317 retrovirus packaging cell lines, and NIH 3T3
thymidine kinase negative cells were grown in
Dulbecco/Vogt-modified Eagle's medium (DMEM) supplemented
with 10$ fetal bovine serum in humidified 5°~ C0~ at 37°C
(Miller et al., 1989, supra, and Miller and Buttimore
Mol. Cell Biol. 6:2895 (1986)). For studies showing the
cell distribution in PTFE implants, LrEpSN transduced
cells were first labeled with the fluorescent marker
1,1'dioctadecyl-3,3,3'3-'tetramethylindocarbocyanine
perchlorate(DiI) as described by Clowes et al., ,1. Clin.
Invest. 93: 699 (1994).
To transduce the cells, early passage smooth
muscle cells were exposed to 16-hr vii-us harvests from
PA317-LrEpSN or PA317-LASN amphotropic virus-producing
cell lines for a period of 29 hr in the presence of
polybrene (4 ~.g/ml). Infected cells were selected ir;
medium containing 6418 at 1 mg/ml. Vascular smooth muscle
cells infected with LrEpSN and selected in 1 mg/ml G-418
antibiotic secreted 5.7 milliunits/29 h per 10~ cells of
Epo.
To achieve cell implantation, two PTFE rings
per animal were positioned under the serosal. plane of the
rat stomach to create an area above the muscle layer
enclosed by the serosa membrane. Figure 1 shows a
schematic cross sectiUn of a PTFE chamber implanted under
the tunica serosa of the stomach. The cross section
shows a view of a pocket which is created by a PTFE ring
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49
when positioned on the muscle layer and seeded with
transduced vascular smooth muscle cells.
Briefly, rats were anesthetized by
intraperitoneal(IP) injection with 94mg/kg ketamine,
5mg/kg xylazine, and 0.5mg/kg acepromazine. All rats
received 0.04 mg dexamethasone IP just prior to surgery.
An area from the thoracic inlet to the pubis was prepared
for surgery and a 3cm m:idline abdominal incision was made
from the xyphoid to the umbilicus. The stomach was
temporarily exteriorized and held in place with a
mosquito hemostat. A 0.5 crn superficial incision was
made in the capsule on the cranial face of the body of
the stomach and a small pocket of approximately 0.6 cm
diameter was created under the capsule using blunt
dissection. A small PTFE ring (inner diameter 4mm, outer
diameter 6 mm) was inserted into the pocket and sutured
in place using 5-0 maxon on a taper needle in a simple
continuous pattern. The suture material was drawn
tightly to constrict the ring to a final inner diameter
of 2 to 3mm before finishing the knot. The fibrous tunic
directly overlying the :ring was cryofrozen using a steel
probe, and the ring was mechanically elevated to prevent
the freezing of the underlying muscular layer to minimize
tissue damage.
Prior to cell implantation, the PTFE ring was
rinsed with 0.9% saline to remove any blood clots formed
during surgery. Cell alz.quots of 1 x lU' cells/50 ml
media were then introduced into the center of the ring
through a 29-g intravenous (IV) catheter. Animals
received two rings each containing 1 x 10" transduced
vascular smooth muscle cells expressing either Epo or
human ADA.
