Note: Descriptions are shown in the official language in which they were submitted.
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A METHOD OF TRANSDUCING MAMMALTAN C1ELLS, AND PRODUCTS
RELATED THERETO
This invention was made with government support
under project number ZO1 AI 00644 and ZO1 AI 00645 funded
by the National Institute o~ Allergy and Infectious
Diseases. The government has certain rights in the
invention.
BACKGROUND OF THE TNVENTTON
The present invention relatE~s generally to
ex v.ivo gene therapy and, more specifically, to an
improved method for transducing cells with viral-vectors.
The introduction of therapeutic genes into
patient cells is a promising approach for the treatment
of human diseases such as inherited genetic disorders,
cancer, infectious diseases and immune' disorders. One
approach for introducing therapeutic genes involves
isolating a target cell population from an .individual,
transfering therapeutic genes into they cells while the
cells are maintained in culture, testing and selecting
for transduced cells, and then reintroducing the
genetically engineered cells into a subject. This
procedure, known as ex vivo gene therapy, is limited by
2S the current inability to achieve high level gene transfer
and expression in clinically relevant numbers of cultured
cells.
Transfer of genes into cells can be
accomplished by a number of physical and biological
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methods. Pure DNA will enter cells following
electroporation or direct microinject:ion, or when the DNA
is complexed with cationic lipids or calcium phosphate.
However, these methods are generally i~oo inefficient and
labor intensive for clinical use.
A more efficient method of gene transfer for
clinical applications involves transduction of cells by
viral-vectors that are genetically engineered to serve as
carriers of heterologous genes. The choice of viral-
vector depends on the target cell type: and transduction
approach deszred. For example, modified adenovirus and
adeno-associated virus can be produced at very high
titers and provide for transiently high levels of gene
expression in target cells. Replication-defective
retroviruses are also used clinically, primarily for
transducing cells where stable integration into the host
chromosomal DNA is desired. The transferred gene is
faithfully replicated and expressed in the progeny of the
retrovirally-transduced cells. Other viruses with
tropisms for specific cell types and engineered hybrid
viruses are also are used in ex vivo gene therapy
applications.
Gene therapy is most beneficial when all or the
majority of cells that are introduced .into a subject
contain the desired genetic modification. However,
current strategies for transducing cells with viral-
vectors have not achieved this goal. One means of
improving transduction efficiency is to bring the
cultured target cells and the viral-vector -into close
proximity. For example, co-cultivation of target cells
with virus-producing cells has been used to achieve high-
efficiency gene transfer. However, co--cultivation raises
concerns about the safety of exposing cells that will be
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introduced into subjects to other cells, as well as
concerns about the reproducibility of infection in such a
co-culture system.
Ex vivo culture and transducaion of cells to
date has generally involved the use of culture plates and
flasks composed of conventional polyst:yrene, which is a
hard, gas-impermeable plastic. Conventional polystyrene
culture vessels are by necessity tied to an open system
for regulation of gases dissolved in t:he culture medium
and for regulation of pH of the cultuz~e medium.
Typically, these conventional culture vessels are
maintained in an incubator filled with regulated
concentrations of OZ and C02; the caps or tops of the
culture vessels must be offset from th.e main vessel, and
thus open to the environment, in order to admit the
ambient gas mixture. As a result, the cell culture is
exposed to contamination from the environment.
Thus, there exists a need for improved methods
fox transducing cells for ex vivo gene therapy
applications. The present invention satisfies this need
and provides related advantages as well.
BRIEF DESCRZpTION OF THE IIJVENTION
In accordance with the present invention, there
is provided a method of transducing ce:Lls comprising
providing a flexible closed culture container having
cells therein and contacting said cell, with a viral-
- vector in the presence of a mufti-functional chemical
moiety. The invention method is useful for efficiently
transducing clinically relevant numbers of mammalian
cells in a closed fluid path system. Also provided are
methods of delivering a functional protein to a subject
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in need thereof, comprising transduc5.ng mammalian cells
according to the invention method and introducing said
cells into a subject in need thereof.
In accordance with another embodiment there is
provided a container system for transducing cells,
comprising a flexible closed culture container and a
mufti-functional chemical moiety therein. The invention
system is useful in the methods described herein for
transducing mammalian cells in a closed fluid path
system.
BRIEF DESCRIPTION OF THF_ FIGURES
Figure 1 shows a top view 1~0 (upper) and side
view 12 (lower) of the flexible culture container 14 not
drawn to scale, which in this example has two ports 16
with connecting tubing 18 to allow slow continuous flow
of liquid medium across the inner sur:Eace 20 to allow
interaction of cells and/or virus veci:.ors with the
surface.
Figure 2 shows a high magnification schematic
of the side view 12 (not drawn to scale) of the flexible
culture container 14 of Figure 1 indi<:ating how target
mammalian cells and virus vector both attach to chemical
moieties displayed at the inner surface 20 of the gas
permeable flexible plastic culture container.
Figure 3 shows the results of a "3-color" flow
cytometric analysis in which. the percE:nt of primitive
cells (CD34 bright, CD38 dim) expressing gpgl~"~X protein
for Patient #1 was assessed as described in Example II.
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Figure 4 shows the results ~of a "3-color" flow
cytometric analysis in which the percent of primitive
cells (CD34 bright, CD38 dim) expressing gp9lPh°x protein
for Patient #2 was assessed as described in Example II.
5 Figure 5 shows the results of a "3-color" flow
cytometric analysis in which the percEent of primitive
cells (CD34 bright, CD38 dim) express_Lng gp9lP''°x protein
for Patient #3 was assessed as described in Example II.
Figure 6 shows an analysis of hydrogen peroxide
production by peripheral blood neutrophils from Patient
#2 using a dihydrorhodamine 123 (DHR) flow cytometric
assay as described in Example II.
DETATZED DESCRIPTION OF THEINVENTION
In accordance with the present invention, there
is provided a method of transducing mammalian cells,
comprising providing a flexible closed, culture container
having cells therein and contacting the cells with a
viral-vector in the presence of a mufti-functional
chemical moiety. By incorporating a m.ulti-functional
chemical moiety into the flexible closed culture
container, the invention method advantageously provides a
deans to increase the viral transduction efficiency of
heterologous genes in a clinically safe environment.
As used herein, the phrase ":mufti-functional
chemical moiety" refers to a substantially pure chemical
moiety having at least one "cell-surface binding domain"
linked to at least one "virus binding .domain" and
optionally linked to other functional domains such as
domains that mediate attachment to a culture container,
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and the like. The multi-functional chemical moiety
functions to colocaiize viral-vectors and target cells,
which results in the increase in transduction efficiency
of the virus relative to the transduci:ion efficiency in
the absence of the multi-functional chemical moiety. The
multi-functional chemical moiety can be attached, by
coating or the like, to the flexible closed culture
containers of the invention, or added to the culture
medium prior to or during viral transduction.
The flexible closed culture container used in
the methods of the present invention provides the
advantages of efficiently transducing clinically relevant
numbers of mammalian cells in a closed fluid path system.
A flexible closed culture container system also
advantageously allows for the culture :media to be
continuously perfused through the container. For
example, it may be useful to gradually add fresh media or
to dilute undesired products without disturbing the cells
cultured therein. By minimizing excessive manipulation
of the cells, their viability and engraftment potential
can be improved. Similarly, the viral supernatant can be
continuously perfused through the container to maximize
the contact of virus with the surface of the flexible
closed container and with the cells therein. If desired,
these solutions can be automatically pumped thxough the
system at a predetermined flow rate.
As used herein, the phrase "flexible closed
culture container" refers to a gas-permeable closed
container with an inner growing surface of polystyrene.
This type of container is exemplified by the PL241?
container Baxter Immunotherapy, Round Lake, IL),
described in PCT US95/13943, which is herein incorporated
by reference in its entirety.
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The inner surface of the flexible container is
preferably an ultra-thin layer of polystyrene having a
thickness of about 0.0004 inches. The outer surface is
composed of a polymer material or a mixture of polymer
materials having a thickness of about 0.006 to about
0.008 inches. Such a container preferably has the
following physical properties: a flexural modulus of
about 10,000-30,000 psi; an oxygen permeability of about
9-15 Barrers; a carbon dioxide permeability of about 40-
80 Barrers; a nitrogen permeability of about 10-100
Barrers: a water vapor transmission rate of not more than
g mi1/100 in2/day; and an optical clarity within the
range of about 0.1% -10% as measured by a Hazometer in
accordance with ASTM D1003. The containers are also
15 capable of withstanding radiation stex°ilization.
For adherent cell culture, t:he growth surface
preferably has a surface energy of greater than about 40
dynes/cm. Most adherent cells require. a negatively
charged growth surface, although some require a
20 positively charged surface. A flexible culture container
for adherent cell culture can therefore have either a
negatively or positively charged surface.
The exchange of 02 and C02 in conventional
tissue culture dishes is not very efficient, particulary
in large culture dishes which could otherwise contain
clinically relevant numbers of cells. One advantage of
the present invention is that the gas ;permeability of the
flexible closed culture container provides for very
efficient exchange of gases. This allows fo.r higher cell
viability and more rapid cell proliferation as compared
to conventional culture containers.
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Conventional, flat tissue culture dishes are
inflexible and, therefore, culture in such dishes
requires a large volume of medium to ensure that the
surface of the dish and the cells thez:eon are covered.
In particular, for the invention tran:~duction procedures,
where it is desired to minimize the volume of viral
supernatant solution in the culture container,
conventional dishes are disadvantageous. The flexibility
of the closed culture container advantageously allows for
the volume of the media or viral supernatant therein to
be minimized. Fox example, the growing surface of the
flexible closed culture container need. only be covered
with a few millimeters of liquid, following which the air
within the container can be sterilely drawn off, in order
to ensure that the cells are sufficiently covered.
In one embodiment, the entire cell collection,
and preselection if desired, is conducted in a closed
fluid path system such as the CS3000/ISOLEX 3001 (Baxter
Immunotherapy, Irvine, CA) which then is aseptically
connected to the flexible culture container for the
transfer of cells into the container. As used herein,
the term "closed fluid path system" refers to an assembly
of components, each of which is closed to the ambient
environment, and each of which is provided with means for
effecting sterile connections among the components. The
multi-functional chemical moiety, the cell suspension,
the viral-vector and the culture media can be added to or
removed from the flexible culture container via sterile
connect ports and sterile tubing systems as set forth in
.Figure I. Alternatively, other configurations of ports
are also contemplated herein in conneci~ion with the
invention flexible culture containers. For example,
there may be only one port with tubing where medium with
cells and/or virus vector enters and exits the same port
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for static incubation and interaction with the
mulifunctional chemical domains at the container's inner
surface. Tubing may be sealed or sterile connected to
pumps and/or reservoir of medium with cells and/or virus
vector.
In addition, samples of traosduced cells can be
aseptically drawn off from the container through sterile-
connect ports for analysis. Following cell transduction,
concentration into an infusible medium such as PLASMA-
LYTE A (Baxter IV Systems, Round Lake,, IL) can be carried
out aseptically via sterile-connect ports, and the washed
and concentrated cells can be infused directly via the
patient's intravenous line without exposing the cells to
the environment, or the personnel to t:he cells.
As used herein, the term "linked" or "linkage,"
when referring to a multi-functional chemical moiety,
refers to an operative connection between a cell-surface
binding domain and a virus-binding domain such that a
target cell and a viral-vector are co-localized for
efficient transduction. Depending on the particular
cell-surface binding domain and virus-binding domain, the
linkage can be accomplished by chemical cross-linking
using agents with twa or more reactive groups at opposite
ends of a linker arm. These cross-linking agents react
with functional groups in the cell surface and virus
binding domain fragments to form stable covalent bonds.
Appropriate cross-linking reagents are knawn in the art
and are readily available commercially.
A mufti-functional chemical moiety that
comprises two or more peptide subunits can also be
recombinantly produced by the expression of a nucleic
acid sequence encoding a cell binding domain and a virus
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binding domain on a single polypeptide~ chain. A multi-
functional chemical moiety can also beg directly
synthesized by methods known in the art.
In one embodiment, a cell-surface binding
5 domain and a virus-binding domain are "linked" by their
independent attachment to the surface of a flexible
culture container, such that the surface of the container
functions as the multi-functional chemical moiety. The
cell-surface binding domain and the virus-binding domain
10 can be attached, either sequentially or together, to the
container so long as each domain is attached to the
surface of the container. Each of the. cell-surface
binding and virus-binding domains are attached to the
surface of the container in sufficient quantity to allow
each domain to be in close enough proximity to each other
to function to co-localize the viral-vectors and cells.
As used herein, the phrase "cell-surface
binding domain" refers to a moiety that has the ability
to attach to the surface of a cell such that domains
other than the cell-surface domain are in close proximity
to the cell surface and available to interact with one or
more other surfaces. The attachment to the cell surface
can be, for example, an attachment to cell membrane
lipids or to proteins or glycoproteins present at the
cell surface. For example, the attacI~ument can be via
binding to receptors on the cell surface, such as
integrins, growth factor and cytokine .receptors, and the
like.
Exemplary cell-surface binding domains are
present in cell-surface binding molecu:Les such as growth
factors and cytokines (eg. , EGF, FGFs, PDGF, insulin,
IGFs, TGFs, VEGF, NGF, G-CSF, GM-CSF, :interferons,
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interleukins, TNFs, and the like, and cell-surface
binding fragments and analogs thereof); extracellular
matrix proteins (eg., fibronectin, collagen, laminin,
vitronectin, thrombospondin, von Willebrand factor,
fibrinogen, tenascin, osteopontin and the like, and cell-
surface binding fragments and analogs thereof);
antibodies and antibody fragments tha?t bind cell surface
molecules; lectins; and cell-cell adhesion molecules
(eg., cadherins, fasciclins and ICAMs" and the like, and
cell binding fragments and analogs thereof).
Particular cell-surface binding domains of
cell-surface binding molecules have besen characterized
and can be used in a method of the invention. For
example, many extracelluar matrix adhEa ion proteins
contain an Arg-Giy-Asp sequence that mediates their
interaction with cell surface integrins (Ruoslahti et
al., Sci~ 238:491-497 (1987)). Pe~>tides including
this sequence are cell-surface binding fragments. Other
cell binding domains in extracellular matrix proteins are
also recognized, such as the TSPN18 and TSPN28
recombinant fragments from the amino terminus of
thrombospondin (Incardona et al., f. Cell. Biochgm.,
62:431-442 (1996)); the collagen peptide motif DGEA
(Matrix Biol., 16:273-283 (1997)); the: pentapeptide YIC;SR
of laminin (Graf et al., Ce , 48:989-996 (1987)), and
the like.
A preferred cell-surface binding domain of the
invention is a cell-surface binding fragment of
fibronectin. Several fragments of fibronectin bind to
integrins on the surface of cells. An exemplary cell-
surface binding fragment of fibronectin is a fragment
containing the sequence Arg-Gly-Asp-Ser (REDS) and
optionally containing the sequence Pro-His-Ser-Arg-Asn
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(PHSRN). Such a fragment may also include all or part of
the ninth and tenth fibronectin Type :CII repeats. A
cell-surface binding fragment of fibronectin can also be
a VLA-4 (a4(31) integrin binding site C:S-1 (connecting
egment-1) within the alternatively spliced IIICS region,
or a cell binding fragment therefrom containing the
tripeptide leu-asp-val (LDV) (Wayner et al., J. Cell
Biol., 116:489-497 (1992)). A further cell binding
fragment of fibronectin is the N-terminal matrix assembly
site that binds VLA-5 (x5(31) integrin (Hocking et al., J .
Cell Biol,s,; 141:241-253 (1998) ) . A cell-surface binding
fragment of the invention can include one or more of
these cell binding domains of fibronectin.
A multi-functional chemical moiety also
includes a virus-binding domain. As used herein, the
term "virus-binding domain"' refers to a moiety that
attaches to the surface of a virus such that domains
other than the virus-binding domain are in close
proximity to the virus surface and available to interact
with one or more other surfaces. Exemplary virus-binding
domains are found in virus-binding molecules such as
virus receptor molecules (eg., CD4, a receptor for HIV;
CR2, a receptor for EBV (Sinha et al., J. Imm n~~ ol.,
150:5311-5320 (1993)); GLVR-2 or Pit2, a receptor for
amphotrophic murine leukemia viruses (wan Zeijl et al.,
Proc. Natl Acad Sci USA, 91:1168-11'72 (1994), Chien et
al., J. Viroloav, 71:4564-4570 (1997)); CAR, a receptor
for coxsackievirus and adenovirus (Bergelson et al.,
Science, 275:1320-1323 (1997)); and the like) and virus-
binding. fragments thereof; antibodies and antibody
fragments that bind virus surface molecules such as env
gene products; heparan sulfate; and heparin binding
domains of extracellular matrix molecu:Les. Another
exemplary virus-binding domain is the high affinity
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heparin-binding domain II of human fibronection (FN type
III repeats 12, I3, 24), and virus-biriding fragments
thereof .
Cell-surface or virus-binding domains can be
modified so long as cell or virus binding activity,
respectively, is maintained. Such modlifications can
include, for example, additions, deletions or
substitutions of natural or non-naturally occuring amino
acids. Structural analogs of natural cell-surface or
virus binding domains, such as peptide mimetics, can also
be produced and are included within the invention, so
long as cell or virus binding activity is maintained.
A cell surface or virus binding domain can be
produced by proteolysis or chemical cleavage of a
molecule containing such a domain and isolation of the
molecule. A cell surface or virus binding domain can
also be recombinantly produced using a:n appropriate
vector containing a nucleic acid sequence encoding such a
domain. A cell surface or virus binding domain can also
be directly synthesized by synthesis methods known in the
art.
A preferred multi-functional moiety suitable
for cell transduction is an extracellu:lar matrix
molecule, or fragment therefrom, that contains both a
cell surface binding domain and a viru~> binding domain.
An exemplary multi-functional moiety is human
fibronectin, whose amino acid sequence is set forth in
.Kornblihtt et al., The EMBO J., 4(7):1755-1759 (1985)
Fibronectin can be purified from plasma, recombinantly
produced, or obtained commercially. "I~iulti-functional
fragments" of fibronectin, which contain one or more
virus-binding and one or more cell-surface binding
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domains of fibronectin, are also inc:Luded within the
invention. These fragments can be produced recambinantly
or by proteolysis of fibronectin.
Mufti-functional fragments of fibronectin
include fragments such as the 30/35 Is:Da carboxy-terminal
chymotryptic fragment of fibronectin (Ruoslahti et al.,
J. Biol. Chem. 256:7277-7281 (1981); Ruoslahti et al.,
Methods Enzymol. 82:803-831 (1982)). Mufti-functional
fragments of fibronectin also include recombinantly
produced fragments such as CH-296, H-296 and CH-272
fragments (Hanenburg et al., Nat,_ ure Med~,.cine 2:876-882}.
The fibronectin fragment CH-296 consists of amino acids
1239 to 1515 recambinantly fused to amino acids 1690 to
1985 of human fibronectin. The fibro;nectin fragment H-
296 consists of amino acids 1690 to 1'985 of human
fibronectin. The fibronectin fragment CH-271 consists of
amino acid 1239 to 1525 recombinantly fused to amino
acids 1690 to 1960 of human fibronectin. The sequences
of fibronectin fragments CH-296, H-29fi and CH-271 are set
forth in U.S. Patent No. 5,198,423, which is incorporated
herein by reference in its entirety.
As used herein, the term "ce:lls" refers to
mammalian ceps capable of being maintained in culture
and of being transduced with viral-vectors. The term is
intended to include both primary cells and established
cell lines. Preferred cells of the invention are human
cells and may be of any relevant tissue origin.
Appropriate target cells for gene transduction include
progenitor cells from various tissues that have the
capacity for self-renewal, including k~eratinocytes,
fibroblasts, hepatocytes and myoblasts. Particularly
preferred cells are human hematopoietic~ cells, such as
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hematopoietic stem cells, lymphocytes, dendritic cells
and monocytes.
The phrase "hematopoietic cells" refers to
cells that are, or differentiate to become, blood and
5 immune cells. Such cells include, for example, stem
cells such as pluripotent hematopoiet:ic stem cells,
including CD39+ stem cells., lymphoid stem cells and
myeloid stem cells. The phrase "hematopoietic cells" as
used herein also includes progenitor and immature cells
10 such as T and B cell progenitors, granulocyte-monocyte
progenitors, eosinophil progenitors, basophil
progenitors, megakaryocytes and erythroid progenitors.
Hematopoietic cells of the invention also include mature
cells such as B and T lymphocytes, de:ndritic cells,
15 monocytes, macrophages, neutrophils, eosinophils and mast
cells. Particularly preferred hematopoietic cells are
lymphocytes, dendritic cells and monocytes.
Hematopoietic cells can be obtained from
various tissue and fluid sources, including, for example,
embryonic yolk sac, fetal liver, spleen, thymus, lymph,
bone marrow, umbilical cord blood and peripheral blood.
A preferred source of hematopoietic cells is peripheral
blood obtained by apheresis. Dendritic cells can also be
obtained from non-lymphoid organs such as the skin.
Transduction of cells by a :method of the
invention can be performed in unselected cell
populations. Alternatively, particular cell types can be
selectively enriched prior to transdu~ction to reduce the
amount of virus required and to increase the transduction
rate relative to unselected populations. Cells that
express a specific protein on their surfaces can readily
be isolated by interaction with a specific binding agent
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such as, for example, a specific antibody. As an
example, CD34+ hematopoietic stem cells can be
selectively enriched prior to transducaion by virtue of
the presence of CD34+ antigen on the surface of these
cells, which can be targeted with a CD34+ monoclonal
antibody. Various means for CD34+ cell selection are
described in the following patent documents: U.S.
5,536,475 U.S. 5,240,856; U.S. 5,411,863, and the like.
Cells that can be transduced by a method of the
invention further include cells that can be
differentiated in culture along a particular cell
lineage, such as dendritic cells. For example,
mononuclear cells can be isolated from a leukapheresis
product by density gradient centrifugation, and cultured
in the presence of hematopoietic growth factors such as
IL-4, GM-CSF, or TNF-a in order to promote the
proliferation and differentiation of dendritic cells.
Transduction by a method of l:.he invention can
be performed in the presence of additives such as dextran
sulfate or "poiycations" that enhance the efficiency of
cell transduction in the presence of a mufti-functional
chemical moiety. As used herein, the germ "polycations"
includes positively charged compositions such as
polycationic lipids, protamine sulfate, poly-L-lysine,
poly-L-arginine, polybrene, chitosan,
poly(ethyleneimine), polymers incorporating basic groups
such as amines, and the like. Appropriate polycations
and concentrations thereof can be determined by one
skilled in the art to maximize transducaion efficiency
and minimize cell toxicity. A preferred polycation is
protamine, which can be used at a concentration of
between 0.5 ug/ml and 10 pg/ml, more preferably at about
6 ug/ml.
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Cell transduction by a method of the invention
can be performed in serum-free, animal protein free
medium. The serum-free, animal protein-free base medium
can be a proprietary medium such as X-VIVO 10 or
X-VIVO I5 (BioWhittaker, Walkersville, MD), Hematopoietic
Stem Cell-SFM media (GibcoBRL, Grand Island, NY) or can
be of any formulation which is favorable to mammalian
cell culture. Serum-free media are described in the
following patent documents: W0 95/00632; U.S. 5,405,772;
PCT US94/09622. The serum-free base medium can contain
clinical grade human serum albumin in a concentration of
about O.S - 5.0%, usually about 1.0% (w/v). Clinical
grade albumin derived from human serum" such as BUMINATE
(Baxter Hyland, Glendale, CA), is so highly purified and'
isolated from other serum components that it is herein
considered serum-free.
Media formulations that are serum-free and
animal protein-free are free from animal proteins such as
those present in fetal calf serum (FCS) and bovine serum
albumin (BSA), which are traditional additives in cell
culture. The absence of animal proteins will abrogate
contact of the cells with a foreign protein which could
detrimentally affect their immune function or otherwise
change their nature. Animal protein-free media
formulations will also abrogate the risk of returning
highly immunogenic animal proteins to the subject, which
could cause anaphylactic shock and death.
As used herein the term "transducing" refers to
introduction of a particular nucleic acid sequence
contained in a "viral-vector" into cells. Transduction
by a method of the invention involves contacting cells
with a viral-vector in the presence of .a multi-functional
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chemical moiety such that the viral nucleic acid enters
the cell and can be expressed therein. Viral based
systems provide the advantage of being' able to introduce
relatively high levels of a heterologous nucleic acid
into a variety of cells.
Suitable viral-vectors for transducing
mammalian cells are well known in the art. These viral-
vectors include, for example, Herpes simplex virus
vectors (eg., U.S. Patent Nos. 5,672,344 and 5,501,979),
Vaccinia virus vectors (eg., Piccini et al., Meth. in
En~S olog,~r, 153: 545-563 ( 1987 ) ) ; Hepat:itis B-based
vectors (eg. Chaisomchit et al., Gene '~er~,
4:1330-1340 (I997)); Cytomegalovirus vectors (eg.,
Mocarski et al . , in V.i~~1 -vectors, Y. (~luzman and S : H.
Hughes, Eds., Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1988, pp. 78-84); Adenovirus vectors (eg.,
U.S. Patent Nos. 5,731,172 and 5,707,618); Adeno-
associated virus vectors (eg., U.S. Patient Nos.
5,741,683; 5,478,745; and 5,756,283); Lentiviral vectors
(eg., Naldini et al., P~oc. Natl Acad Sci. USA,
93:11382-11388 (1996)); Parvovirus vectors (eg., U.S.
Patent No. 5,585,254); Retrovirus vectors (eg., U.S.
Patent Nos. 4,405,?12; 4,650,764; 5,686,279; 5,591,624;
5, 693, 508; 5, 747, 323; 5, 667, 998; ; 5, 672, 510; 5, 580, 766
and 5,716,826), and the like.
Particularly preferred viral-vectors for a
method of the invention are replication-defective
retroviral-vectors. Genes transferred .in retrovirai
vectors are integrated into chromosomal DNA and are
therefore faithfully replicated and expressed in progeny
cells. These vectors are capable of mediating stable
gene integration in a wide range of target cell types
without unacceptable gene deletion or gene rearrangement
CA 02340086 2001-02-09
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19
in the host genome. Replication-defective retroviral
vectors generally do not contain gag, poZ or env gene
sequences, and are incapable of produc:~ng progeny virus
after transduction of the target cell. Replication-
defective retroviral vectors can be derived from
amphotrophic viruses, such as the Moloney Murine Leukemia
Virus (Mo-MuLV). An especially prefers°ed viral-vector is
the MFGS retrovirus vector (available ~:rom Cell Genesys,
Foster City, CA; Weil et al.', Blood. 89:1754-1761
(1997)).
Retroviral vectors are packaged in special
"packaging" cell lines that are genetically modified to
produce the gag, pol and env proteins required to make a
complete virus particle. The gene of interest is
inserted into the retrovirus vector DNP,, which is
transfected into a packaging line to obtain clones of
cells that package and shed recombinant replication-
defective retrovirus. Packaging cell lines for
replication-defective retroviruses can contain CRIP
packaging plasmids, such as the ~r-CRIP NIH 3T3 rnurine
fibroblast line (Danos et al., Proc. Natl. Acad. Sci.
UPS , 85:6460-6464 (1988)). A preferred packaging cell
line is the amphotropic 293-SPA human embryonic kidney
cell packaging line (Davis et al., Hum. Gene Therapy,
8:1459-1467 (1997)).
Viral-vectors of the invention can also be
packaged for the production of pseudotype viruses. Such
hybrid viruses contain the nucleic acid and certain
protein components of one type.of virus enclosed in the
capsid or outer coat of a different types of virus.
Pseudotype viruses are advantageous in combining useful
features of two or more viruses. For example, a viral
nucleic acid that is particularly amenable for cloning or
CA 02340086 2001-02-09
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for replication within a target cell can be packaged
within a viral coat that provides fo:r enhanced robustness
or a particular cell tropism. A pari~icular example of a
pseudotype virus is a retroviral veci~or packaged with the
5 vesicular stomatitis virus-G (VSV-G) envelope.
In accordance with another embodiment of the
present invention, there is provided a container system
for transducing cells, comprising a flexible closed
culture container and a mufti-functic>nal chemical moiety
10 therein. A flexible closed culture container having a
mufti-functional chemical moiety therein can be prepared,
for example, in an aqueous buffer such as phosphate-
buffered saline (PBS). The solution containing the
mufti-functional chemical moiety can be applied to the
15 flexible culture container in sufficient volume to cover
the growing surface of the container, and incubated at
room temperature or at 37°C for several hours. For
coating a flexible culture container, an appropriate
concentration of mufti-functional chemical moiety is
20 between about 1 ug/ml and 1 mg/ml, preferably between
about 10 ug/ml and 200 ug/ml. The solution can
optionally be removed following incubation.
Alternatively, the mufti-functional chemical moiety can
be added to the cell culture medium o:r the viral
supernatant preceding or during the t:ransduction
procedure.
In accordance with yet another embodiment of
the present invention, there is provided a method of
delivering a functional.prote'in to a subject in need
thereof, comprising transducing cells within a flexible
closed culture container having cells therein, according
to a method of the present invention, and introducing the
transduced cells into a subject in need thereof.
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21
Typically, a subject in need thereof is a
patient having a "pathology," which i.s an abnormal
disease state characterized by, for example, genetic
defects, infection, neoplasia, altered immune function,
tissue damage, and the like. Thus, in accordance with
the invention methods of delivering functional proteins
to subjects in need thereof, a pathology can be treated
by introducing cells transduced with appropriate genes
into a patient. Far example, a genetic deficiency can be
ZO treated by introducing into a subject cells transduced
with nucleic acid sequences that encode a wild-type copy
of a defective gene. These nucleic acid sequences
express proteins, such as structural proteins or enzymes;
that compensate for the genetic deficiency. Therapeutic
genes need not be delivered to the ce:Ll type that is
itself affected. For example, cells i~hat are engineered
to express and secrete various enzymes can be reimplanted
in various areas of the body to produce secreted
products.
Representative genetic deficiencies that can be
treated by a method of the invention include, for
example, severe combined immunodeficie:ncy caused by
adenosine deaminase (ADA) deficiency or purine nucleoside
phosphorylase (PNP) deficiency, chronic granulomatous
disease (CGD) caused by phox subunit gene deficiency,
diabetes caused by insulin deficiency,
hypercholesterolemia caused by LDL receptor deficiency,
hemophilia caused by Factor IX deficiency, and
thalassemias and anemias caused by globin gene
deficiencies.
Neoplasias can also be treated by a method of
the invention. Genetically modified cells can be
introduced that express a gene that contributes to the
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22
inhibiton of neoplastic cell growth . Such genes can be
tumor suppressors such as p53 and Rb; encode anti-sense
or ribozyme molecules that inhibit oncogene expression;
induce drug sensitivity to tumors; increase the immune
response to tumors; or encode cytotox.ic products. For
example, cells can be transduced with Fas ligand for the
killing of Fas-expressing tumors. Tumor cells can also
be transduced with genes that make them sensitive to
killing by the subsequent administration of a
chemotherapeutic drug. For example, the herpes virus
thymidine kinase (HSVTK) can be introduced into tumor
cells and the patients treated systemically with
gancyclovir. A "bystander effect" provides killing of
both transduced and non-transduced tumor cells. Tumor
I5 cells can also be transduced with cyt~okine genes, such as
TL-2 and GM-CSF, and injected into sulbjects as vaccines
to enhance anti-tumor immune responses.
Proteins or peptide fragmenits thereof that are
characteristic of particular cell types can be recognized
by the immune system when appropriately presented by
antigen-presenting cells. For examples, a cell can be
transduced with a tumor-specific molecule and returned to
the individual to initiate humoral and cell-mediated
immune responses for the elimination of neoplastic cells
bearing that molecule. Similarly, proteins or peptides
that are characteristic of cells infecaed by viruses and
other pathogens can be appropriately ~>resented to the
immune system following transduction Hrith a viral-vector
encoding the characteristic protein or peptide.
The toxicity of many chemoth.erapeutic agents is
due to myeloablation with concomitant leukopenia and/or
thrombocytopenia, and creating a population of
hematopoietic stem cells that are resistant to specific
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23
drugs is advantageous. Therefore, normal cells,
particularly normal hematopoietic stem cells, can be
transduced with genes that confer re~~istance to
chemotherapeutic agents as an adjunct: to traditional
cancer therapy. For example, patient. cells can be
transduced with genes encoding multiple drug resistance
(such as MDR and MRP), dihydrofolate reductase,
methylguanine methyltransferase, aldehyde dehydrogenase
and the like, and increased doses of chemotherapeutic
agents can be given.
Disorders of the immune system that can be
treated by a method of the invention include autoimmune
disorders. Numerous disorders are believed to result
from autoimmune mechanisms, including, for example,
rheumatoid arthritis, multiple sclerosis, type I
diabetes, systemic lupus erythematosua, myasthenia
gravis, psoriasis and pemphigus vulgaris. Immune cells
can be transduced with autoantigens which are
appropriately presented to the host irnmune system to
induce tolerance to the autoantigen, thereby treating the
autoimmune disease.
Disorders of the immune sy~~tem that can be
treated by a method of the invention also include
disorders caused by rejection of allog~eneic cells or
tissues. Allogeneic MHC molecules are highly immunogenic
and will trigger rejection of the grafted cells if there
are too many mismatches. Therefore, rejection of
allogeneic cells can be prevented by transducing the
allogeneic cells with MHC molecules that match the
recipient or with molecules that block the mismatched MHC
molecules.
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24
Infectious disorders can b~e treated by
introducing cells into a subject that have been
transduced with genes that inhibit viral infection,
replication or assembly. Such genes encode, for example,
anti-viral antibodies, mutant viral :receptors that
prevent viral attachment, anti-viral ribozymes, anti-
sense transcripts, and the like. For example, AIDS and
HIV-1 pathologies can be treated or prevented by
tranducing hematopoietic stem cells o~ith dominant-
negative mutant of HIV gag, rev or iut linked to an HIV
promoter. The differentiated cells derived from this
transduced population are protected from HIV infection.
Similar strategies could also be employed to treat or
prevent other infectious diseases.
Cardiovascular diseases are also amenable to
gene therapy: For example, the introduction of cells
transduced with a gene encoding lecithin: cholesterol
acyltransferase can reduce atherosclerosis; and the genes
encoding nitric oxide synthase, Fas ligand and GAX
(growth arrest-specific homeobox) delivered to
endothelial tissues can prevent or treat restenosis.
Pathologies that can be tre<~ted by a method of
the invention particularly include disorders of the
hematopoietic system. Hematopoietic cells are
straightforward to isolate and contain populations of
stem cells that can divide and differentiate to
repopulate the hematopoietic system. As used herein the
term "hematopoietic disorder" include:> disorders
. 30 affecting hematopoietic cells, includ.i.ng: neoplasias such
as Hodgkin's and non-Hodgkin's B and T cell lymphomas and
leukemias: genetic disorders such as t.halassemias,
Fanconi anemia; adenosine deaminase (1~,DA) deficiency, X-
linked severe combined immunodeficiency syndrome (X-
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SCID), chronic granulomatous disease (CGD), Gaucher's
disease, Hurler's syndrome, sickle cE:ll disease and the
likes infectious disorders such as AI:DS~ as well as
hematopoietic and immune dysfunction~~ caused by
5 chemotherapy and radiation treatment.
Chronic granulomatous disease (CGD) is an
inherited deficiency of the immune system caused by the
failure of blood neutrophil leukocyte, to produce
microbicidal hydrogen peroxide. CGD patient blood
10 neutrophils have , defect in the NADP~H oxidase enzyme
required for production of superoxide, the chemical
precursor of hydrogen peroxide. The phagocyte NADPH
oxidase is composed of several protein subunits. A
genetic mutation affecting production of any one of the
15 subunits results in the patient having CGD. The two most
common forms of CGD are the X-linked variety resulting
from defects in the gene encoding the gp9lph°" oxidase
subunit protein and an autosomal recessive variety
resulting from defects in the gene encoding the p47p''°x
20 oxidase subunit protein. Since hemat~apoietic CD34+ stem
cells give rise to the circulating blood neutrophils, the
invention methods have been employed to permit CGD
patient CD34+ stem cells to produce the missing oxidase
subunit could correct the oxidase defect of CGD because
25 functionally corrected neutrophils ar<s produced from the
gene corrected stem cells by proliferation and
differentiation.
Cells that have been transduced by a method of
the invention are introduced into a subject in need
thereof to effectively treat the pathology. The
transduced cells can be cells previous>ly obtained from
the same individual or an MHC-matched individual.
Introduction of cells into a subject i.s generally
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26
accomplished by intravenous infusion of cells in an
infusion solution. A preferred infu~;ion solution is
PLASMALYTE infusion solution (available from Baxter
HealthCare) containing to human serum albumin.
The invention will now be f~escribed in greater
detail by reference to the following non-limiting
examples. All U.S: patents and all publications
mentioned herein are incorporated in their entirety by
reference thereto.
Z~ Exam~I
~,nsduct~on of CD34+ Cells in Fi~exibl_e Conta~ne_rs
'n P
.ial ~;hemical Moiety
This example demonstrates that human cells can
be efficiently transduced in flexible closed culture
containers in the presence of a multi~-functional chemical
moiety.
The efficiencies of transducing human
hematopoietic stem cells in fibronect:in fragment-coated
and uncoated flexible closed culture containers and
conventional culture plates were compared. Specifically,
the following four culture container <:onditions were
compared: 1) a fibronectin fragment-coated gas-permeable
flexible plastic containers 2) an uncoated gas-permeable
flexible plastic container; 3) a fibronectin fragment-
coated conventional hard plastic tissue culture plate
and 4) an uncoated conventional hard plastic tissue
culture plate, The multifunctional chemical moiety used
to coat the surface of the culture ve~~sel was a
recombinant human carboxyl-terminal fragment of
fibronectin designated CH-296 (available from
BioWhittaker Co.; see U.S. Patent 5,198,423). The Baxter
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27
PL2417 gas-permeable flexible plastic: culture containers
used were the 120 ml sized (15 cm x T.5 cm surface area
per side holding 120 ml fully inflate:d).
The multi-functional chemical moiety CH-296 was
dissolved in phosphate buffered saline with added HEPES
buffer (PBS at pH 7.2-7.4) at a concentration of
100 ug/ml and 10 ml of this solution introduced into the
PL2417 container using a 22 gauge needle and 2O cc
syringe. Overnight incubation at room temperature
allowed the CH-296 fibronectin fragment to coat the inner
surface of the PL2417 container by electrostatic
attraction. At that time the CH-296 fibronectin fragment
solution was removed and the containers were blocked for
30 min with 15 ml of 2% bovine serum albumin. The
containers were then washed three times with 40 ml of
Hank's buffered saline solution (HBSS;! containing HEPES
buffer. The 6-well conventional hard plastic tissue
culture plates (35 mm diameter wells) were similarly
treated with 5 ml of the solution of ~?BS with fibronectin
fragment CH-296 to allow coating of the surface, followed
by blocking and washing. As controls, uncoated PL2417
flexible gas-permeable containers or Ei-well conventional
tissue culture plates were similarly x>locked and washed.
While the above-described conditions were used
in the initial experiments, subsequent studies have
demonstrated similar results can be obtained: 1.) when
coating with PBS solutions without HEPES, but containing
20 to 100 ug/ml CH-296; 2.) when the coating times are 2
hours to overnight; and 3.) when the blocking solution is
1% human serum albumin (HSA) in PBS instead of 2% bovine
serum albumin in HBSS with HEPES.
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Initial transduction efficiency experiments
were performed with human CD34+ hemat:opoietic stem cell
cells obtained from the peripheral b7_ood of normal donors
that had been treated with 5 or 6 daily doses of
granulocyte colony stimulating factor (G-CSF, Amgen) to
mobilize stem cells from the peripheral blood. In
subsequent experiments, the peripheral blood CD34+ stem
cells were similarly obtained from patients with the
inherited immune deficiency known as X-linked gp9lp''~X-
deficient chronic granulomatous disease. Volunteers or
patients were subjected to an apheres~is procedure on day
5 or 6 of G-CSF mobilization to harvest a mononuclear
cell fraction enriched for the stem cells using the
CS3000 Plus blood cell separatar device (Baxter
Healthcare, Fenwal Division, Deerfield, IL). This
apheresis product was subjected to an antibody selection
procedure using monoclonal antibody directed at the CD34+
antigen following manufacturer's instructions using the
ISOLEX 300 SA immunomagnetic stem cell selection system
device (Baxter Healthcare, Fenwal Division, Deerfield,
IL). The purified CD34+ cells were cryopreserved in l0a
DMSO/90~ fetal bovine serum in the vapor phase over
liquid nitrogen until use.
For the initial studies of transduction
efficiency of CD34+ cells in the fibr~onectin fragment
coated PL2417 flexible gas-permeable .containers, the
MFGS-gp9lph~" amphotropic retrovirus vector was used. The
MFGS retrovirus vector backbone has been described
previously and has been used in a variety of clinical
gene therapy settings (see, e.g., Malech et al. ~NAS. USA
94:22133-12138, 1997) The retrovirus vector was produced
using the 293 SPA packaging cell line" which is a human
embryonic kidney cell line engineered to produce the
virus packaging proteins: gag, pol and amphotropic
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29
envelope (see Davis et al., Human Gene Theraov 8:1459-
1467, 1997) . The MFGS-gp9lp''~" retrov:irus vector was
collected from confluent producer cell layers of the 293-
SPA amphotropic packaging line over a~ 12 hour period in
X-VIVC7 10 medium (a serum-free, animal protein tree
medium available from BioWhittaker) supplemented with la
human serum albumin (HSA) and is designated as Vector
Supernatant (VSN). This VSN is the vector used in all of
the examples/experiments described below.
In particular, the MFGS-gp9lp''~" vector contains
the coding sequence of human gp9ip''~", one of the subunits
of the phagocytic blood cell NADPH oxidase enzyme (see
Li, F, et al. Blood 84:53, 1994). This vector was
specifically designed for clinical application for gene
therapy to treat the gp9lp''~x protein dleficient form of the
inherited immune deficiency known as :K-linked chronic
granulomatous disease. It is also a convenient marker
gene because an FITC conjugated anti-gp9lph~x monoclonal
antibody (antibody 7D5) can be used in a flow cytometric
assay to detect expression of this therapeutic protein as
a marker at the surface of cells succE~ssfully transduced
with this retrovirus vector.
It is known that patients with X-linked CGD
have a genetic defect resulting in failure of mature
differentiated phagocytic cells (e.g. neutrophils) to
produce gpglp''~x and therefore fail to generate the
microbicidal oxidants, superoxide and hydrogen peroxide.
MFGS-gp9lph~x amphotropic retrovirus transduction of CD34+
cells from patients with gp9lph°'~.-deficient X-linked CGD
with MFGS-gp9lpho" retrovirus vector can be assessed
either by using flow cytometry to detect gp9lp~'~"
expression in the cultured CD34+ cells or by using a flow
cytometry assay of oxidase activation (oxidant
CA 02340086 2001-02-09
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production) in neutrophils differentiated from transduced
CD34+ cells. However, even in transciuced normal CD34+
cells the early expression of gp9lP''°" detected by antibody
labelling and flow cytometry can be used as a marker to
5 indicate successful transduction by this retrovirus
vector. This is because this protein is produced only
later in differentiation of myeloid cells and is normally
absent from the surface of even normal CD34+ cells during
the first 9 or 10 days in culture.
10 In the afternoon of experimental "day 0" frozen
normal human CD34+ cells (obtained and stored as noted
above) were thawed and resuspended in 15 ml of X-VIVO 10
medium supplemented with 1% HSA and the following human
recombinant growth factors: (10 ug/ml G-CSF, 100 pg/ml
15 Pixykine [interleukin 3/granulocyte colony stimulating
factor {GM-CSF}fusion protein from Immunex Corp], 100
pg/ml flt3-ligand [flt3L from Immunex Corp], and 100
ug/ml stem cell factor [SCF from R&D .Systems]). This
medium is designated in this example and other examples
20 as "standard CD34+ cell medium." The cells with medium
were incubated in a standard tissue culture flask
overnight in a 37° C tissue culture incubator at 10% C02.
The next morning (experimental "day 1"') CD34+ cells were
spun down by low speed centrifugation and suspended in
25 transduction medium consisting of 50% standard CD34+ cell
medium and 50% VSN (i.e. MFGS-gp9lPh°" vector supernatant
harvested by the 293-SPA packaging line as above) to
which was added 6 ug/ml clinical grade: protamine. For
the portion of the experiment using tree 120 ml sized
30 PL2417 flexible containers, 20 ml of t:he transduction
medium with 3.5 x 10 6 CD34+ cells was placed in each of
two flexible containers (fibronectin fragment CH-296
coated versus uncoated). For compari~;on 5 ml of the
transduction medium with 0.5 x 106 cells was placed in
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31
each of two wells (fibronectin fragm~;nt CH-296 coated
versus uncoated) of a 6-well standard tissue culture
plate.
After 6 hrs of the "day 1" transduction, the
contents of the flexible containers or the wells of the
plates were removed and the 4 samples individually spun
down to recover the cells while discarding the
transduction medium. The cells were resuspended in a
volume of standard CD34+ cell medium overnight equal to
the original volume (5 ml for the well samples and 20 ml
for the flexible container samples) and returned to the
original culture vessel for an overnight incubation. The
next morning the contents of each vessel were removed and
centrifuged, the cells resuspended in the same respective
volumes of transduction medium, and the cells with
transduction medium returned to the respective original
culture vessels for the "day 2" trans~duction of 6 hrs.
At the end of the "day 2" transductio:n, the cells were
handled exactly as for "day 1" wherethey were removed
from the transduction medium and resu;spended in the
original volume of standard CD34+ cel:Z medium for
overnight incubation in the respective= original vessels.
The transduction pracess was repeated on "day 3" and then
the cells were left in standard CD34+ cell medium until
analysis on "day 6." All transductions, overnight
cultures and subsequent post-transduci:ion culture took
place in a 37° C tissue culture incubator at 10% C02.
On "day 6" the transduced CI)34+ cells were
washed and labelled with FITC-conjugated anti-human
gp9lph°" monoclonal antibody. Fluores~~ence flow
cytometric analysis revealed that 82% of the cells in the
fibronectin fragment CH-296 coated PL2417 flexible
culture container expressed the recom~>inant gp9lp''°X, while
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32
only 270 of the cells in the uncoated PL2417 flexible
culture container expressed the recombinant gpglph~".
Thus, because of the inherent uncertainty of conducting
transfections in flexible, plastic, gas-permeable culture
containers, there was a surprising 3-fold increase in the
transduction rate in the fibronectin coated versus
uncoated flexible culture container indicating the
importance of fibronectin for efficient transduction.
Similarly, 86% of cells cultured in t:he fibronectin
fragment coated well of a six well plate expressed the
recombinant gp9lP''~x and this represented a more than 2
fold increase of transduction rate co3:npared to that seen
with the uncoated well.
Several subsequent studies using the same
flexible containers and conditions demonstrated that
coating of the PL2417 flexible containers with
fibronectin fragment CH-296 consisteni=ly resulted in
transduction rates that were 3 to 7 fold higher than the
results seen in uncoated bags. Similar results of
fibronectin fragment enhanced transducer ion were obtained
when the clinically applicable larger 1 liter sized
PL2417 flexible containers (17 cm x 22.5 cm were studied)
were coated with fibronectin fragment. In those
laboratory studies the 1 liter sized bag was coated for 2
to 6 hours at room temperature with 4CI ml of PBS
containing 20 ug/ml fibronectin fragment CH-296 and
washed 3 times with 60 ml of PBS containing to clinical
grade HSA.
.With the conditions noted in. the detailed
experiment above, the inclusion of protamine in the
transduction medium surprisingly resulted in maximum
transduction. Without inclusion of protamine during the
transduction period, the transduction enhancing effect of
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33
coating the vessel with fibronectin fragment was markedly
reduced. For example; in two experiments with
fibronectin coated vessels conducted where conditions
were identical to the experiments noted in detail above
except for the inclusion. or exclusion of 6 ug/ml
protamine in the transduction medium transduction rates
were 71~ with protamine versus 53~ wi.thout protamine in
the first experiment and 90o with protamine versus 160
without protamine in the second experiment. Thus,
inclusion of protamine in the liquid phase of the
transduction medium unexpectedly demonstrated synergy
with the effect of coating the culture vessel with
fibronectin fragment CH-296 in enhancing transduction in
the PL2417 flexible plastic containers.
is Examx~l_e II
Eli-n_i_~al_ Trial
The following examples demonstrate that patient
cells transduced ex vivo in gas-permeable flexible
culture containers in the presence of a multi-functional
chemical moiety can be used in gene therapy applications.
Since the preclinical studiEas outlined in
Example I indicated that coating of the PL2417 flexible
containers with the mufti-functional <:hemical moiety
corresponding to the fibronectin fragnnent CH-296 could
greatly enhance retrovirus transduction, the~invention
method was employed to conduct a clinical trial of gene
therapy for the X-linked, gp9lp''°" defi~~ient form of CGD.
This clinical trial is currently ongoing and the results
of this trial have not been published.
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34
Three patients with the X-Linked gp9lph~"
deficient form of CGD were administered 8 daily
subcutaneous doses of the marrow growth factors
Granulocyte-Macrophage Colony Stimulating Factor 5 ug/kg
plus flt3L 50 ~Zg/ml (both growth factors were obtained
from Immunex Corp.). On day 8 and 9, the patients
underwent an apheresis procedure processing 15 liters on
the CS3000 blood cell separator as outlined in Example I.
CD34+ cells were separated~from the apheresis products
using the fully automated ISOLEX 3001 immunomagnetic
processor (Nexell Therapeutics Inc., Irvine, CA)
following manufacturer's instructions. The separated
CD34+ cells, which averaged 80~ to 90~ purity and
numbered from 150 to 300 x 106 cells total from each
apheresis preparation, were immediately suspended in
about 120 mI of clinical CD34+ cell medium (defined as X-
VIVO 10 medium supplemented with 1o HSA and the following
human recombinant growth factors: 50 ~.~g/ml Pixykine
[Immunex Corp), 100 ug/ml flt3L [Tmmunex Corp], and 50
ug/ml SCF [R&D Systems]. Thus, the clinical CD34+ cell
medium was very similar to the standard CD34+ cell medium
described in Exam lp a I except for somES minor changes in
the concentrations of the growth factors and exclusion of
G-CSF.
~ The cells were incubated overnight in a
fibronectin fragment CH-296 coated 1 Liter size PL2417
flexible plastic container (coating was performed as in
the last paragraph of Example ~). The: next morning a
small aliquot of cells was set aside for culture without
transduction to serve as a negative ce~ntrol for later
analysis of transduction efficiency. For the bulk of the
cells, aliquots of 100-200 x 106 patient CD34+ cells were
centrifuged and re-suspended in about 120 ml of clinical
transduction medium prepared similar to that described in
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Exa,~nole T except that the ratio of VSN and clinical CD34+
cell medium was 90:10 and the growth factor content of
the final clinical transduction medium was identical to
that in clinical CD34+ cell transduction medium. Each
5 120 mi aliquot of cells was placed in a fibronectin
fragment CH-296 coated 1 liter sized PL2417 container and
incubated for 6-7 hrs to allow transduction to occur.
Using this volume in this sized flexible container
advantagously results in a~fluid layer that is only 3 to
10 4 mm in depth maximizing interaction of the retrovirus
vector and cells with the inner coated surfaces of the
PL2417 container. At the end of the transduction, the
contents of each bag were removed and pooled, centrifuged
and resuspended in an equal volume of clinical CD34+ cell
15 medium. The CD34+ cells derived from the second
apheresis from the same patient were 'then pooled with the
cells from the first apheresis that h,ad undergone a
single cycle of transduction. The 120 ml each of the
pooled cells were returned to the coated PL2417 bags that
20 had been used for the tranduction and incubated
overnight. The next morning the cello were counted,
centrifuged and resuspended in aliquoits of 100-200 x 106
cells in about 220 ml of clinical transduction medium.
Each 120 ml aliquot was placed into a fibronectin
25 fragment CH-296 coated PL2417 flexiblE: plastic container
and incubated for 6-7 hrs as with the first day
transduction. Except that no additional fresh cells were
added to the pool, the process was repeated on a third
and fourth day.
30 At the end of the fourth transduction a small
aliquot of the transduced cells was centrifuged,
resuspended in standard CD34+ cell medium and set aside
for additional culture to allow later analysis of
transduction efficiency and assessment; of functional
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36
correction of neutrophils differentiated in culture from
the transduced patient CD34+ stem cells: The bulk of the
transduced autologous CD34+ stem cells were washed
several times in Plasmalyte A (Baxter, Hyland Division)
containing 1% HSA and resuspended into about 50 ml of
this fluid in a sterile 60 ml syringe. After safety
tests to assure sterility, the cells were administered
intravenously to the respective patients.
As in EXamp a I above, transduction efficiency
as determined by flow cytometric analysis of surface
expression of recombinant gp9lPh°" was determined two to
three days after the last transduction. Analysis was
preformed on the small aliquots of tr~ansduced CD34+ cells
that had been set aside in culture from the bulk of the
cells that had been administered to tike patients as gene
therapy treatment. This was compared to the sample of
the same patient's cells that had been set aside for
culture without undergoing the transduction procedure. A
slightly more complex flow cytometric analysis was
performed in order assess the efficiency of transduction
of the phenotypically more primitive item cells. It is
known that the group of cells that express high levels of
surface CD34 antigen and iow levels oi: CD38 antigen
contain the bulk of the colony forming cells and
primitive stem cells.
By labelling the transduced or non-transduced
patient CD34+ cells with antibodies to CD34 antigen, CD38
antigen and gp9lph°", each with a distinct fluorescent
probe it is possible to perform a "3-c;olor'° flow
cytometric analysis in which the percent of primitive
cells (CD34 bright, CD38 dim) expressing gp9lPh°X protein
can be assessed. This analysis is shown in Figure 3 for
patient #l, Figure 4 for patient #2 anal Figure 5 for
CA 02340086 2001-02-09
WO 00109168 PCT/US9811b699
37
patient #3. For each figure four dot plot panels are
shown. In each case the left two panels are the non-
transduced cells and the right two panels are the
clinically prepared transduced cells. In the upper
panels the analysis shows the CD38 (vertical axis) and
CD34 (horizontal axis expression by these cells). Those
cells in the small rectangle seen at the lower right
corner of the cluster of cells are those phenotypically
primitive cells analyzed for gp9lP''°" F,rotein expression.
This analysis of this selected group ~of primitive cells
is shown in the lower panels. The dot plots in the lower
panels plot cell size (side scatter) on the vertical axis
versus gp9lPh°X expression on the horizontal axis. Cells
to the right of the vertical line in ithe lower panels are
I5 defined as gp9lp''°X positive. In the non-transduced
population this line is set to allow <~ background of
about 2.2% positivity.
By this analysis, the transduction of cells
from patient #1 was 82.9% (after subtracting background):
from patient #2 was 80.3%; and from patient #3 was 62.0%.
These clinical scale transduction rate's are at least
about 3 to 5 times higher than that rE:ported in Malech et
al. (1997), supra, and also many time; higher than any
reported experience with transduction of human CD34+
cells in the clinical setting. Furthermore, there was an
average 3.5 fold expansion of the CD34+ cells over the 4
days of the clinical scale transduction regimen such that
the final harvest of CD34+ cells was from 1 to 2 x 109
cells at over 92% viability. In addition the cell
3U products were well tolerated by the patients and. passed
all FDA mandated safety testing. These data demonstrate
that fibronectin fragment CH-296 coated PL2417 flexible
plastic containers can be used in the clinical setting of
ex vivo gene therapy to achieve extraordinarily high
CA 02340086 2001-02-09
WO 00/09168 PCT/US98/16699
38
transduction rates of phenotypically primitive stem cells
while at the same time fostering proliferation and
survival of those cells, and yielding a cellular
composition fit for intravenous administration for
treatment of patients.
Preliminary results are available indicating
engraftment of these transduced cells in the CGD
patients. In particular it is shown :in Figure 6 that 25
days after intravenous administration of these transduced
cells.in Patient #2 functionally normal oxidase positive
neutrophils could be detected in the ~?eripheral blood.
Figure 6 is an analysis of hydrogen pE~roxide production
by peripheral blood neutrophils from i~his patient using a
dihydrorhodamine 123 (DHR) flow cytomEaric assay. The
35 details of this assay have been descr~Lbed or referred in
detail in Malech, HL, et al: Proc. Natl. Acad. Sci. USA
94:12233-12138, 1997. In brief, peripheral blood is
drawn, the red cells lysed to obtain t:he blood leukocytes
which are then loaded with the DHR ester for 5 minutes.
The DHR is de-esterified and thus traX>ped in the
neutrophils. The neutrophils are therk stimulated to
produce hydrogen peroxide by exposure to phorbol
myristate acetate. If oxidants are produced, then the
DHR is oxidized with resultant increase in its
fluorescence. CGD oxidase negative neutrophils
demonstrate low fluorescence while normal oxidase
positive neutrophils exhibit high fluorescence in this
assay. The appearance of small numbers of high
fluorescence neutrophils in a CGD patient following gene
therapy is evidence of the production of.functionally
normal neutrophils which must be derived from gene-
corrected CD34+ cells that have engrafted in the
patient's bone marrow.
CA 02340086 2001-02-09
WO 00/09168 PCT/US98/16699
39
The three panels of Figure 6 show dot plots in
which cells with the size characteristics (side scatter
on the vertical axis) of neutrophils are analyzed for
oxidant production (DHR fluorescence in the horizontal
axis). The top panel shows neutraphils from the
peripheral blood of a normal volunteer where almost all
the neutrophils are highly fluorescent (e.g., far to the
right of the vertical line defining positivity). The
middle panel shows this analysis of peripheral blood
neutrophils from patient #2 prior to gene therapy where
all of the cells show low fluorescence. indicating lack of
oxidase activity. At 25 days after the administration of
the transduced CD34+ cells almost 1 in 1000 of his
peripheral blood neutrophil demonstrate high fluorescence
equal to normal cells indicating high oxidase activity in
these gene therapy corrected neutrophi.ls. Thus, the use
of fibronectin fragment CH-296 coated PL2417 bags for
enhanced ex vivo transduction of CD34+ cells yielded a
cell composition that was capable of e:ngraftment and
capable of mediating production of functionally corrected
neutrophils into the peripheral blood of a treated
patient.
While the invention has been described in
detail with reference to certain preferred embodiments
thereof, i.t will be understood that modifications and
variations are within the spirit and mope of that which
is described and claimed.
