Note: Descriptions are shown in the official language in which they were submitted.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
DESCRIPTION
HUMAN FIBROBLAST GROWTH FACTOR RECEPTOR 1 IS A CO
RECEPTOR FOR INFECTION BY ADENO-ASSOCIATED VIRUS 2
BACKGROUND OF THE INVENTION
The present application claims the benefit of U.S. Provisional Patent
Application, Serial Number 60/114,596, filed December 31, 1998. The government
may own rights in the present invention pursuant to Public Health Service
grant
numbers HL-48342, HL-53586, HL-58881, and DK-49218, from the National
Institutes of Health (Centers of Excellence in Molecular Hematology).
1. Field of the Invention
The present invention relates generally to the fields of gene therapy. More
particularly, it concerns gene transfer using adeno-associated virus and
methods of
increasing transcription and promoting replication of transgenes.
2. Description of Related Art
Gene therapy protocols involving recombinant viral vectors are gaining wide
attention as a new weapon against disease. Of the different viral vectors used
for gene
transfer, the retrovirus and adenovirus-based vector systems have been the
most
extensively investigated. Recently, adeno-associated virus (AAV) has emerged
as a
potential alternative to the more commonly used retroviral and adenoviral
vectors
(Muzyczka, 1992; Carter, 1992; Flotte and Carter, 1995; Chatterjee et al.,
1995;
Chatterjee and Wong, 1996). While studies with retroviral- and adenoviral-
mediated
gene transfer raise concerns over potential oncogenic properties of the
former, and
immunogenic problems associated with the latter, AAV has not been associated
with
any such pathological indications (Berns and Bohenzky, 1987; Berns and Giraud,
1996).
The viral genome of AAV is a single-stranded DNA of 4,680 nucleotides,
flanked at both ends by 145 nucleotide-long palindromic inverted terminal
repeats
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
2
(TTRs) (Srivastava et al., 1983). Wild-type AAV has been shown to integrate
into
human chromosome in a site-specific manner (Kotin et al., 1991; Samulski et
al.,
1991), whereas recombinant AAV vectors appear not to integrate site-
specifically
(Kearns et al., 1996; Ponnazhagan et al., 1997). In previous studies, it has
been
documented that transduction efficiency of AAV vectors in permissive cells
correlates
well with the phosphorylation state of a cellular protein, designated the
single-
stranded D-sequence-binding protein (ssD-BP), which preferentially interacts
with the
D(-) sequence within the AAV TTR sequence (Qing et al., 1997; Qing et al.,
1998; and
U.S. Patent application serial number 09/145,379, specifically incorporated
herein by
reference in its entirety). Two independent groups have presented evidence
suggesting that following infection, the rate-limiting step for the efficient
transduction
by AAV is the viral second-strand DNA synthesis (Fisher et al., 1996; Ferrari
et al.,
1996). Further, it has been demonstrated that the tyrosine phosphorylation
state of the
ssD-BP correlates well with the efficiency of AAV-mediated transgene
expression in
vivo as well (Qing et al., 1998).
AAV possesses a broad host-range that transcends the species barrier
(Muzyczka, 1992). Recently, cell surface heparan sulfate proteoglycan (HSPG)
was
identified as a receptor for AAV (Summerford and Samulski, 1998). The
ubiquitous
expression of HSPG, perhaps, accounts for the broad host-range of AAV.
However, it
also has become increasingly clear that, for the most part, efficient viral
infection of
the host cell is accomplished in at least two steps: attachment and entry,
presumably
requiring at least two distinct cell surface macromolecules, a receptor and a
co-
receptor, respectively. A cogent example of such an event has been presented
in the
context of infection by the human immunodeficiency virus 1 (HIV 1 ) which
utilizes
the cell surface CD4 antigen as a site of attachment followed by one of the
chemokine
receptors for the viral entry (Alkhatib et al., 1996; He et al., 1997).
Similar scenarios
also have emerged for efficient infection by adenovirus and herpesvirus
(Laquerre et
al., 1998; Bergelson et al., 1997; Montgomery et al., 1996; Geraghty et al.,
1998).
Improving the efficiency of AAV infection will increase the use of this vector
in gene therapy applications. However, to date the identity of a receptor that
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
3
successfully mediates infection of cells by AAV has remained elusive. Once
such a receptor
or factor is elucidated, it will be possible to increase the efficiency of AAV-
mediated
gene therapy.
SUMMARY OF THE INVENTION
The present invention, for the first time, describes the co-receptor needed
for
AAV entry into a cell. In a particular embodiment of the present invention,
there is
described a method of increasing AAV infection of a cell comprising increasing
the
amount of fibroblast growth factor receptor (FGFR) on the surface of the cell,
wherein
the increased FGFR increases the AAV uptake by the cell. By increasing the
amount
of FGFR of said cell, the present invention refers to any method that
effectively
increases the number of accessible AAV binding sites on the surface of said
cell.
Thus it is envisioned that such sites may be made available through
engineering steps
or through the application of agents that allow for a change in protein
confirmation
such that the AAV-binding sites become exposed to the AAV being presented.
In specific embodiments, increasing the amount of FGFR on the cell may
comprise providing to the cell an expression construct comprising a
polynucleotide
encoding an FGFR polypeptide and a promoter active in eukaryotic cells, the
polynucleotide being operably linked to the promoter. In preferred
embodiments, the
FGFR polypeptide is selected from the group consisting of FGFR1, FGFR2, FGFR3
or FGFR4. In other preferred embodiments, it is contemplated that the method
further
comprises increasing the amount of cell surface heparan sulphate proteoglycan
(HSPG) on the cell. In particular embodiments, increasing the HSPG of the cell
comprises providing to the cell an expression construct comprising a
polynucleotide
that encodes an HSPG polypeptide and a promoter active in eukaryotic cells,
the
polynucleotide being operably linked to the promoter. In still other
embodiments, it
may be possible to increase the AAV binding capacity of the HSPG receptor by
altering it conformation and/or glycosylation pattern.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
4
In certain embodiments, the method further may comprise contacting the cell
with an AAV vector. In more particular embodiments, the AAV vector is a vector
comprising an expression cassette comprising a selected polynucleotide and a
promoter active in eukaryotic cells, wherein the polynucleotide is operably
linked to
the promoter. More specifically, it is contemplated that the selected
polynucleotide
encodes a polypeptide. In other embodiments, the selected polynucleotide may
encode an antisense construct. In yet another alternative, the selected
polynucleotide
encodes a ribozyme. In defined embodiments, the promoter may be any promoter
known to be useful in a particular gene delivery application. In certain
embodiments,
the promoter may be an inducible promoter. In certain preferred embodiments,
the
promoter is CMV IE, SV40 IE, HSV tk, (3-actin, human globin a,, human globin
(3,
human globin y, RSV, B19p6, alpha-1 antitrypsin, PGK, tetracyclin, MMTV or
albumin promoter.
In specific embodiments, of the present invention, the expression cassettes)
further may comprise a polyadenylation signal. More particularly, the
polyadenylation
signal may be an AAV polyadenylation signal, an SV40 polyadenylation signal or
a
BGH polyadenylation signal. Of course these are merely exemplary
polyadenylation
signals, it is understood that those of skill in the art will be able to
substitute other
polyadenylation signals therefor and arrive at an expression cassette that may
be used
in the method of the present invention.
In specific embodiments, the selected polypeptide may be a hormone, a tumor
suppressor, an inhibitor of apoptosis, a toxin, a lymphokine, a growth factor,
an
enzyme, a DNA binding protein or a single-chain antibody. In those embodiments
in
which the present invention provides a receptor encoding expression construct,
the
expression construct may be a viral vector. More particularly, the viral
vector may be
selected from the group consisting of retrovirus, adenovirus, vaccinia virus,
herpesvirus and adeno-associated virus.
The present invention further provides a method of expressing a selected
polynucleotide from an adeno-associated viral (AAV) vector in a host cell
comprising
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
the steps of providing an AAV vector comprising an expression cassette
comprising
the selected polynucleotide and a promoter active in eukaryotic cells, wherein
the
selected polynucleotide is operably linked to the promoter; contacting the
vector with
the host cell under conditions permitting uptake of the vector by the host
cell; and
5 increasing the amount of fibroblast growth factor receptor (FGFR) on the
surface of
said cell; wherein the increased FGFR increases the uptake of AAV by the cell.
This
increased uptake of AAV will thereby increase the level of transcription of
the
selected polynucleotide increased relative to the transcription of the
selected
polynucleotide in a cell where FGFR activity is not increased. Similarly, by
providing
the cell with an increased ability for AAV infectivity, the methods of the
present
invention will increase AAV-mediated transduction efficiency of a selected
polynucleotide in a host cell.
In specific embodiments, the method of increasing FGFR on the surface of the
cell comprises providing to the cell an expression construct comprising a
polynucleotide encoding an FGFR polypeptide and a promoter active in
eukaryotic
cells, the polynucleotide being operably linked to the promoter. In preferred
embodiments, the FGFR polypeptide is selected from the group consisting of
FGFRl,
FGFR2, FGFR3 or FGFR4. The method further may comprise increasing the amount
of cell surface heparan sulphate proteoglycan (HSPG) in the cell. More
particularly,
increasing the HSPG of the cell comprises providing to the cell an expression
construct comprising a polynucleotide that encodes an HSPG polypeptide and a
promoter active in eukaryotic cells, the polynucleotide being operably linked
to the
promoter. In certain embodiments, the HSPG encoding polynucleotide and the
FGFR
encoding polynucleotide are in the same expression construct. In other
embodiments,
the HSPG encoding polynucleotide and the FGFR encoding polynucleotide are
separated by an IRES. It is contemplated that the HSPG encoding polynucleotide
and
the FGFR encoding polynucleotide each may be under the control of a separate
promoter operative in eukaryotic cells.
It is contemplated that the methods of the present invention may be carried
out
on any cell amenable to gene therapy and/or delivery manipulations. In
particular
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
6
embodiments, the host cell is an erythroid cell. In other defined embodiments,
the
erythroid cell is a human erythroid cell. In certain embodiments, the host
cell may be
selected from the group consisting of a bone marrow cell, a peripheral blood
cell, a
lung cell, a gastrointestinal cell, an endothelial cell and myocardial cell.
In specific
embodiments, the host cell is in an animal.
In particular embodiments, the method further may comprise inhibiting the
function of D sequence binding protein (D-BP) in the host cell. More
particularly, the
inhibiting may comprise reducing the expression of D-BP in the host cell. In
other
embodiments, reducing the expression of D-BP may be achieved by contacting the
host cell with an antisense D-BP polynucleotide. More particularly, the
antisense D-
BP polynucleotide may target a translational start site. In other embodiments,
the
antisense D-BP polynucleotide targets a splice junction site. In certain
embodiments,
the agent that reduces the expression of D-BP is an antibody or a small
molecule
inhibitor. In specific embodiments, the antibody may be a single chain
antibody or a
monoclonal antibody. In other embodiments, the inhibiting comprise reducing
the D
sequence binding activity of the D-BP in the host cell. In specific
embodiments,
reducing the binding activity is achieved by inhibiting the tyrosine
phosphorylation of
D-BP. More particularly, inhibiting the phosphorylation is achieved by
contacting the
host cell with a D-BP peptide containing a tyrosine residue. In specific
embodiments,
inhibiting the phosphorylation is achieved by contacting the host cell with an
agent
that inhibits tyrosine kinase. In defined embodiments, the tyrosine kinase is
an EGF-
R tyrosine kinase. In specific embodiments, the agent is an inhibitor of EGF-R
that
reduces the expression of EGF-R protein kinase. In other embodiments, the
inhibitor
of EGF-R protein kinase is an agent that binds to and inactivates EGF-R
protein
kinase. In still further embodiments, the inhibitor of EGF-R protein kinase
inhibits
the interaction of EGF-R with a D-BP. In specific aspects, the agent that
reduces the
expression of EGF-R protein kinase is an antisense construct, in other
aspects, the
agent that binds to and inactivates EGF-R protein kinase is an antibody or a
small
molecule inhibitor. In particularly preferred embodiments, the agent may be
selected
from the group consisting of hydroxyurea, genistein, tyrphostin l, tyrphostin
23,
tyrphostin 63, tyrphostin 25, tyrphostin 46, and tyrphostin 47.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
7
Another aspect of the present invention provides a method for providing a
therapeutic polypeptide to a cell comprising the steps of providing an AAV
vector
comprising an expression construct comprising the a polynucleotide that
encodes the
polypeptide and a promoter active in eukaryotic cells, wherein the
polynucleotide is
operably linked to the promoter; contacting the vector with the cell under
conditions
permitting uptake of the vector by the cell; and increasing the amount of
fibroblast
growth factor receptor (FGFR) on the surface of the cell; wherein the increase
in
FGFR results in an increase in the uptake of the vector by the cell. In
preferred
embodiments, the FGFR polypeptide is selected from the group consisting of
FGFRI,
FGFR2, FGFR3 or FGFR4. In particular embodiments, the therapeutic polypeptide
is
a hormone, a tumor suppressor, an inhibitor of apoptosis, a toxin, a
lymphokine, a
growth factor, an enzyme, a DNA binding protein or a single-chain antibody. In
certain preferred embodiments, the cell is located within a mammal. In
particular
embodiments, the cell is a cancer cell. More particularly, the cell is
selected from the
group consisting of lung, breast, melanoma, colon, renal, testicular, ovarian,
lung,
prostate, hepatic, germ cancer, epithelial, prostate, head and neck,
pancreatic cancer,
glioblastoma, astrocytoma, oligodendroglioma, ependymomas, neurofibrosarcoma,
meningia, liver, spleen, lymph node, small intestine, blood cells, colon,
stomach,
thyroid, endometrium, prostate, skin, esophagus, bone marrow and blood.
Thus in a broad sense the present invention provides a method for treating a
disease in a subject comprising the steps of providing an AAV vector
comprising an
expression cassette comprising a therapeutic polynucleotide and a promoter
active in
eukaryotic cells, wherein the therapeutic polynucleotide is operably linked to
the
promoter; contacting the vector with the host cell under conditions permitting
uptake
of the vector by the host cell; and increasing the amount of FGFR on the
surface of
said cell; wherein the increase in FGFR on the cell surface increases the
ability of said
cell take up AAV. Thus the AAV is able to provide the therapeutic
polynucleotide to
a cell of said subject where the polynucleotide is transcribed and effects a
treatment of
the disease. The disease may be any disease that can be treated by the
application of a
polynucleotide e.g., cystic fibrosis, cancer, hyperproliferative disorders and
the like.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
8
Also provided herein is an adenoassociated viral expression construct
comprising: a first polynucleotide encoding a selected gene and a first
promoter
functional in eukaryotic cells wherein the polynucleotide is under
transcriptional
control of the first promoter; and a second polynucleotide encoding an FGFR.
In
preferred embodiments, the FGFR polypeptide is selected from the group
consisting
of FGFR1, FGFR2, FGFR3 or FGFR4. In specific embodiments, the expression
construct further may comprise a third polynucleotide encoding an HSPG
polypeptide.
In preferred embodiments, the FGFR encoding polynucleotide is under the
control of
the first promoter. In other embodiments, the first polynucleotide and the
second
polynucleotide are separated by an IRES. In specific embodiments, the second
polynucleotide is under the control of a second promoter operative in
eukaryotic cells.
In particularly preferred embodiments, the selected gene encodes a protein
selected
from the group consisting of a tumor suppressor, a cytokine, a receptor,
inducer of
apoptosis, and differentiating agents. In those embodiments in which the gene
encodes a tumor suppressor, the tumor suppressor may selected from the group
consisting of p53, p16, p21, MMACl, p73, zacl, C-CAM, BRCAI and Rb. In those
embodiments in which the gene encodes an inducer of apoptosis, the inducer of
apoptosis may be selected from the group consisting of Harakiri, Ad E1B and an
ICE-
CED3 protease. In those embodiments where the gene encodes a cytokine, the
cytokine is selected from the group consisting of IL,-2, IL-2, IL-3, IL,-4, IL-
5, IL-6, IL-
7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL.-15, TNF, GMCSF, (3-
interferon
and 'y-interferon. In those embodiments, in which the gene encodes a receptor
other
that FGFR and HSPG, the receptor may be selected from the group consisting of
CFTR, EGFR, VEGFR, IL-2 receptor and the estrogen receptor.
The present invention further contemplates a pharmaceutical composition
comprising a first adenoassociated viral expression construct comprising a
promoter
functional in eukaryotic cells and a first polynucleotide encoding a selected
polypeptide, wherein the first polynucleotide is under transcriptional control
of the
promoter; a second polynucleotide encoding an FGFR; and a pharmaceutically
acceptable buffer, solvent or diluent. In specific embodiments, the
composition
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
9
further may comprise a second expression construct comprising a third
polynucleotide
encoding an HSPG polypeptide wherein the third polynucleotide operatively
linked to
a third promoter.
Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however,
that the detailed description and the specific examples, while indicating
preferred
embodiments of the invention, are given by way of illustration only, since
various
changes and modifications within the spirit and scope of the invention will
become
apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included
to further demonstrate certain aspects of the present invention. The invention
may be
better understood by reference to one or more of these drawings in combination
with
the detailed description of specific embodiments presented herein.
FIG. lA-FIG. lE: Analysis of binding (FIG. lA) of wt AAV to different cell
types, and comparative analyses of transduction efficiency (FIG. 1B-FIG. lE)
of the
recombinant vCMVp-lacZ vector in HeLa and NIH3T3 cells. Equivalent numbers of
human KB, HeLa, 293, M07e, and murine NIH3T3 cells were analyzed in binding
assays in triplicate using S-AAV as described in Methods. Approximately
equivalent
numbers of HeLa (FIG. 1B and FIG. 1C) and NIH3T3 (FIG. 1D and FIG. lE) cells
25 were either mock-treated (FIG. 1B and FIG. 1D), or treated with 500 mM of
tyrphostin 1 for 2 h (FIG. 1 C and FIG. lE), and infected with 2x 103
particles/cell of
vCMVp-lacZ under identical conditions. Forty eight hours post infection
(p.i.), cells
were fixed, stained with X-gal and photographed using a Nikon inverted light
microscope. Magnification x 100.
FIG. 2A and FIG. 2B: Comparative analyses of binding of 'ZSI-bFGF (FIG.
2A) and 35S-AAV (FIG. 2B) to M07e and Raji cells following either mock-
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
transfection, or stable transfection with HSPG and/or FGFR 1 expression
plasmids.
Approximately equivalent numbers of cells were analyzed in triplicate as
described in
Example 1.
5 FIG. 3A-FIG. 3H: Analysis of transgene expression in M07e (FIG. 3A-FIG.
3D) and Raji (FIG. 3E-FIG. 3H) cells. Equivalent numbers of mock-transfected
or
stably transfected cells with the indicated expression plasmids were infected
with 104
pa.rticles/cell of the vCMVp-lacZ vector under identical conditions and
analyzed by
FACS 48 h p.i. as described in Methods. For each sample, 1x104 cells were
analyzed.
10 The percentages of cells in the M 1 region expressing the transgene are
provided in
Table 3.
FIG. 4A-FIG. 4F: Comparative analyses of transgene expression in M07e
(FIG. 4A-FIG. 4C) and Raji (FIG. 4D-FIG. 4F) cells stably co-transfected with
HSPG+FGFR1 expression plasmids in the presence of co-infection with adenovirus
or
with prior treatment with tyrphostin 1. Equivalent numbers cells were infected
with
104 particles/ml of the recombinant vCMVp-lacZ vector under identical
conditions.
Forty-eight hours post infection, cells were analyzed by FACS as described in
the
legend to FIG. 3. The data in FIG. 4A and FIG. 4D indicate mock-transduced
cells.
For each sample, 1 x 104 cells were analyzed. The percentages of cells in the
M 1
region expressing the transgene are provided in Table 4.
FIG. SA-FIG. SH: Effect of bFGF and EGF on AAV binding to non-
permissive (FIG. SA) and permissive (FIG. SB) cells, and on entry (FIG. SC-
FIG. SH)
into permissive cells. 35S-AAV binding assays were caxried out with equivalent
numbers of NIH3T3 and 293 cells essentially as described in the legend to FIG.
1
except that large excess of either heparin (6 mg), bFGF (6 mg), EGF (6 mg), or
unlabeled wtAAV ( 1 x 10'° particles), were included in the reaction
mixtures.
Equivalent numbers of 293 cells also were used to infect with 2x 103
particles/cell of
the recombinant vCMVp-lacZ vector with no treatment (a), with prior treatment
with
150 mM genistein (b), in the presence of similar excess of bFGF (c), with
prior
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
11
treatment with 150 mM genistein and bFGF (d), in the presence of similar
excess of
EGF (e), or with prior treatment with 150 mM genistein and EGF (f). Cells were
fixed, stained, and photographed as described in the legend to FIG. 1.
Magnification
x 40.
FIG. 6A-FIG. 6B: A model for the role of cell surface HSPG and FGFR1 in
mediating AAV binding and entry into the host cell. Co-expression of HSPG and
FGFR1 is required for successful binding of AAV followed by viral entry into a
susceptible cell (FIG. 6A), both of which are perturbed by the ligand, bFGF,
which
also requires the HSPG-FGFR1 interaction (FIG. 6B).
FIG. 7: AAV co-receptor activity of FGFR1, FGFR2, FGFR3, and FGFR4.
These assays were performed as described in Example 1.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The use of viral vectors in a variety of gene transfer endeavors now is widely
accepted. For example, retroviral vectors have been used for a number of years
to
transform cell lines in vitro for the purpose of expressing exogenous
polypeptides.
More recently, with advancements in genetic therapies. various other vectors
including adenoviruses and herpesviruses, along with retroviruses, and more
recently
adeno-associated viruses, have been utilized to transfer therapeutic genes
into cells.
While retroviral vectors and adenoviral vectors have been associated with a
wide variety of pathological indications, adeno-associated viral (AAV) vectors
are
considered especially desirable for a number of reasons. In the first
instance, AAVs
are not associated with any known pathological indications. Further, AAV can
infect
non-dividing cells (Kotin et al., 1990; Kotin et al., 1991; Samulski et al.,
1991) and
also possesses anti-oncogenic properties (Berns and Giraud, 1996). Recombinant
AAV vectors can be produced that lack any of the coding sequences of wild-type
AAV, yet retain the property of stable chromosomal integration and expression
of the
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
12
recombinant genes upon transduction both in vitro and in vivo (Bertran et al.,
1996;
Kearns et al., 1996; Ponnazhagan et al., 1997a).
Despite the fact that AAVs clearly are an attractive alternative to other
viral
vectors, the use AAV as a delivery vector has been limited. The efficiency of
AAV
infection of cells is low even though AAV possesses a broad host-range that
transcends inter-species barriers (Muzyczka, 1992). One factor suggested to
explain
the broad host range is that the cell surface heparan sulfate proteoglycan
(HSPG) may
be a receptor for AAV (Summerford and Samulski, 1998). However, the inventors'
recent studies have documented a significant donor variation in terms of the
ability of
AAV vectors to transduce primary human bone marrow-derived CD34+ hematopoietic
progenitor cells (Ponnazhagan et al., 1997). It was demonstrated that AAV
failed to
bind to CD34+ cells from approximately 50% of normal volunteer donors.
Nonetheless, the lack of virus binding to cells was insufficient to account
for the
inability of AAV to infect cells. For example, in our preliminary experiments
we
noted that murine NIH3T3 cells could bind the virus efficiently, but could not
be
transduced by AAV. Thus, the inventors set out to look for a putative cell
surface co-
receptor for efficient infection by AAV. The present invention is directed to
the
elucidation of a co-receptor for infection by AAV. Methods and compositions
relating to this finding are described in further detail herein below.
A. The Present Invention
Although the cell surface heparan sulfate proteoglycan (HSPG) has been
identified as the putative receptor for AAV infection (Summerford and
Samulski,
1998), it seems that the transduction efficiency of AAV vectors varies greatly
in
different cells and tissues in vitro and in vivo. However, in summarizing
their
findings, Summerford and Samulski, stated that HSPG is only initial attachment
receptor for AAV-2, and that it is possible that AAV attachment and infection
can also
be mediated by an as yet unidentified receptor. Furthermore they suggested
that their
studies were able to show the HSPG is required for AAV infection but were not
able
to show whether HSPG is sufficient for infection. The present inventors have
conclusively shown that cells which express the HSPG receptor alone are unable
to be
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
13
infected by AAV. The inventors reasoned that although HSPG may be responsible
for
AAV binding to cells, AAV entry into the cells is mediated by another
receptor.
The present invention shows that cell surface expression of HSPG alone is
insufficient for AAV infection, and that AAV also requires human fibroblast
growth
factor receptor (FGFR) as a co-receptor for successful viral entry into the
host cell.
The inventors document that cells that do not express either HSPG or FGFR fail
to
bind AAV, and consequently, are resistant to infection by AAV. These non-
permissive cells are successfully transduced by AAV vectors following stable
transfections with cDNAs encoding the murine HSPG and the human FGFR.
Furthermore, AAV infection of permissive cells, known to express both FGFR and
the
epidermal growth factor receptor (EGFR), is abrogated by treatment of cells
with
basic fibroblast growth factor (bFGF), but not with epidermal growth factor
(EGF).
Briefly, the present inventors were able to demonstrate that two non-
permissive human cells, M07e and Raji, can be transduced with AAV following
introduction of two genes: murine HSPG and human FGFR1. It was interesting to
note that with Raji cells, expression of either the HSPG or the FGFR gene was
insufficient to permit AAV binding and entry. In M07e cells, on the other
hand,
introduction of the HSPG gene was sufficient to allow AAV to bind and enter
these
cells. Upon closer examination, it was noted that M07e cells express an
endogenous
FGFR gene. Thus, successful AAV binding and subsequent entry requires co-
expression of both HSPG and FGFR, much the same way as the FGF ligand
(Rapraeger et al., 1991, Ledoux et al., 1992, Roghani and Moscatelli, 1992,
Givol and
Yayon, 1992, Kan et al., 1993). Interestingly, however, despite similar levels
of co-
expression of HSPG and FGFRI in M07e and Raji cells, as determined by the bFGF
binding, the AAV transduction efficiency in the two cell types was
significantly
different. Further, AAV binding to the two cell types also was roughly the
same, but
the extent of viral DNA entry correlated well with the transduction efficiency
in the
two cell types. It remains possible, therefore, that other cellular factors
are required
for high-efficiency infection by AAV (Mah et al., 1998).
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
14
It is known that NIH3T3 cells express both the endogenous HSPG and the
FGFR genes, and that AAV could indeed bind to the muHSPG-muFGFR complex.
Since AAV failed to gain entry into these cells, it would seem reasonable to
suggest
that the specificity of viral entry lies with the huFGFR. Two additional sets
of data
corroborate this contention. First, the muHSPG gene is functional in human
cells, and
second, huFGF abrogates AAV binding as well as entry into otherwise permissive
human cells. EGF, on the other hand, has no effect on either AAV binding or
entry
into 293 cells which express high numbers of EGFR (Mah et al., 1998). Thus,
the
lack of effect of EGF on AAV-mediated transduction of 293 cells is not due to
the
absence of EGFR in these cells. HuFGF, which can bind to muFGFR, also is able
to
block AAV binding to NIH3T3 cells. Additional studies carried out with NIH3T3
cells stably transfected with the huFGFRI expression plasmid resulted in an
increase
in AAV transduction efficiency, albeit at low-levels, most likely due either
to
suboptimal cell surface expression of the human protein, or some form of
steric
hindrance with the murine counterpart. Based on all the available information,
we
propose a model for AAV infection, which is depicted in FIG. 6. In this model,
co-
expression of cell surface HSPG and FGFR1 is required for successful AAV
binding
followed by viral entry (FIG. 6A), both of which are blocked by bFGF (FIG.
6B).
Given these findings, it now is possible to envision the improved use of AAV
vectors in human gene therapy. For example, ensuring that the cells about to
receive
AAV mediated gene therapy express FGFR and HSPG will ensure an efficient
binding
and uptake of the AAV and will thereby increase the effectiveness of the
therapy
being applied. Methods and compositions for achieving such improved gene
therapy
are described in greater detail herein below.
B. Receptors for AAV infection
The inventors have identified a co-receptor responsible for efficient AAV
infection. Recently, HSPG was identified as a putative receptor for AAV
(Summerford and Samulski, 1998). However, it is clear that efficient viral
infection
of the host cell by AAV is accomplished in at least two steps-- attachment and
entry--
presumably requiring at least two distinct cell surface macromolecules, a
receptor and
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
a co-receptor, respectively. The present inventors have identified FGFR as the
essential co-receptor necessary for AAV infection. These two receptors and
their
roles in AAV infection are discussed in further detail herein below.
5 Although it was recently suggested that HSPG is a receptor for AAV
infection,
murine NIH3T3 which are know to express HSPG, failed to be infected by AAV as
discussed above. The present invention describes the putative co-receptor for
AAV
infection. The present inventors investigations, described in the example,
revealed
that this co-receptor is FGFR (Rapraeger et al., 1991; Ledoux et al., 1992;
Roghani
10 and Moscatelli, 1992; Givol and Yayon, 1992; Kan et al., 1993). The present
invention describes a model for AAV infection (FIG. 6). In this model, co-
expression
of cell surface HSPG and FGFR 1 is required for successful AAV binding
followed by
viral entry (FIG. 6A), both of which are blocked by bFGF (FIG. 6B).
15 a. FGF Receptors
Fibroblast growth factors (FGFs) regulate a diverse range of physic logic
processes such as cell growth and differentiation and pathologic processes
involving
angiogenesis, wound healing and cancer (Basilico and Moscatelli, 1992). FGFs
utilize a receptor system to activate signal transduction pathways (Klagsbrun
and
Baird, 1991; Ornitz et al., 1992; Yayon et al., 1991; Rapraeger et al., 1991).
The
primary component of this system is a family of signal-transducing FGF
receptors
(FGFRs). FGFRs are typical of polypeptide growth factor receptors. These
receptors
usually have three major identifiable regions. The first is an extracellular
region
which contains the domain that binds the polypeptide growth factor (i.e. the
ligand-
binding domain). The second region is a transmembrane region and the third is
an
intracellular region. Many of these receptors contain a tyrosine kinase domain
in the
intracellular region. It is contemplated that according to the present
invention, FGFR
family members may act as co-receptors for AAV infection.
The FGFRs contain an extracellular ligand-binding domain and an
intracellular tyrosine kinase domain (Basilico and Moscatelli, 1992). The
second
component of this receptor system consists of heparan sulfate (HS)
proteoglycans or
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
16
related heparin-like molecules which are required in order for FGF to bind to
and
activate the FGFR (Ornitz et al., 1992; Yayon et al., 1991). Although the
mechanism
by which heparin/HS activates FGF is unknown, heparin, FGF and the FGFR can
form a trimolecular complex (Ornitz et al., 1992). Heparin/HS may interact
directly
with the FGFR linking it to FGF (Kan et al., 1993). Furthermore, heparin/HS
can
facilitate the oligomerization of two or more FGF molecules, which may be
important
for receptor dimerization and activation (Ornitz et al., 1992).
Heparin/HS is a heterogeneously sulfated glycosaminoglycan that consists of a
repeating disaccharide unit of hexuronic acid and D-glucosamine. It has been
previously reported that, at a minimum, highly sulfated octa- (Ornitz et al.,
1992) or
decasaccharide (Ishihara et al., 1993) fragments derived from heparin are
required for
FGF to bind to the FGFR.
The fibroblast growth factor receptor (FGF-R) proteins bind to a family of
related growth factor ligands, the fibroblast growth factor (FGF) family. This
family
of growth factors are characterized by amino acid sequence homology, heparin-
binding avidity, the ability to promote angiogenesis and mitogenic activity
toward
cells of epithelial, mesenchymal and neural origin.
The FGF family includes acidic FGF (aFGF) and basic FGF (bFGF)
(Gospodarowicz et al., 1986); the int-2 gene product (Moore et al., 1986); the
hst gene
product or Kaposi's sarcoma FGF (Anderson et al., 1988; Taira et al., 1987);
FGF-5
(Zhan et al., 1988); and keratinocyte growth factor (Rubin et al., 1989), and
FGF-6 (I.
Marics, et al., 1989). The actions of these FGFs are mediated through binding
to
specific high affinity cell surface receptors of approximately 145 and 125 kDa
(Neufeld and Gospodarowicz, 1986; U.S. Patent 5,733,893 ). U.S. Patents
5,707,632;
5,229,501 and 5,783,683 (each specifically incorporated herein by reference)
describe
methods and compositions relating to the identification and purification of
various
fibroblast growth factor (FGF) receptors.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
17
Although FGFRs have been shown to be expressed in every organ and tissue
examined (Givol and Yayon, 1992), the relative abundance of their expression
in
skeletal muscle and in neuroblasts and glioblasts in the brain correlates
particularly
well with the documented high efficiency of AAV-mediated transduction in these
tissues in vivo (Qing et al., 1998). Since there are at least four distinct
but related
members in the FGFR family --FGFR1 (Genbank Accession Nos. P11362; U23445;
U22324, each specifically incorporated herein by reference), FGFR2 (Genbank
Accession Nos. P21802; L49241; L49240; L49239; L4923, each specifically
incorporated herein by reference), FGFR3 (Genbank Accession Nos. P22607;
AF055074; Q61851, each specifically incorporated herein by reference), and
FGFR4
(Genbank Accession Nos. AF031695; Q03142; P22455, each specifically
incorporated herein by reference) -- it is likely that these individual
members may be
useful alone or in combination with each other in facilitating successful
infection by
AAV (Rapraeger et al., 1991, Ledoux et al., 1992, Roghani and Moscatelli,
1992,
Givol and Yayon, 1992, Kan et al., 1993, Lee et al., 1989).
Four genes encode the four forms of FGFR1-4, which have a common
structure composed of two or three extracellular immunoglobulin (Ig)-like
loops (IgI-
IgllI) and one intracellular tyrosine kinase domain. For FGFR1-3, alternative
splicing
of the exon encoding the extracellular region produces multiple receptor forms
(Johnson et al., 1991 Givol and Yayon, 1992). The genomic organization of the
third
Ig-like loop leads to three receptor variants. Two membrane-spanning forms are
produced by alternative splicing of two exons (IIIb and IIZc) encoding the
second half
of loop III, whereas a selective polyadenylation site preceding exons IIIb and
IIIc is
used to produce a soluble form of FGFR1 (IIIa). In humans and mice, the mRNA
transcript of the IgllIa splice variant of FGFR1 encodes a protein that
potentially has
no hydrophobic membrane-spanning domain and may therefore be a secreted form
of
the receptor (SR) (Werner et al., 1992). Those of skill in the art are
referred to
Guillonneau et al., (1998, specifically incorporated herein by reference),
which
provides a comprehensive discussion about the various FGFR isoforms.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
18
b. HSPG Receptors
It is well documented that heparan sulphate proteoglycans (HSPGs) play
important biological roles in cell-matrix adhesion processes and are essential
regulators (or receptors) of growth factor actions. Proteoglycans are proteins
classified by a posttranslational attachment of polysaccharide
glycosaminoglycan
(GAG) moieties each comprised of repeating disaccharide units (for reviews see
references Jackson et al., 1991; Kjellen and Lindahl, 1991). They can be found
associated with both the extracellular matrix and plasma membranes.
The four main, widely distributed, membrane-associated GAGs include
heparin/HS and chondroitin sulfates A through C. These unbranched sulfated
GAGS
are defined by the repeating disaccharide units that comprise their chains; by
their
specific sites of sulfation, and by their susceptibility to bacterial enzymes
known to
cleave distinct GAG linkages (Linhardt et al., 1986). All have various degrees
of
sulfation which result in a high density of negative charge. Proteoglycans can
be
modified by more than one type of GAG and have a diversity of functions,
including
roles in cellular adhesion, differentiation, and growth. In addition, cell
surface
proteoglycans are known to act as cellular receptors for some bacteria and
several
animal viruses (Rostand and Esko, 1997), including; foot-and-mouth disease
type O
virus (Jackson et al., 1996), HSV types 1 and 2 (Sheik et al., 1992; WuDunn
and
Spear, 1989) and dengue virus (Chen et al., 1997).
Summerford and Samulski (1998) recently showed that HSPGs serve as a
principal attachment receptor for AAV type 2 (AAV-2). Further, their results
indicate
that the presence of HS GAG on the cell surface directly correlates with the
efficiency
by which AAV can infect cells. Several observations led Samulski and
Summerford
to postulate that AAV-2 may use cell surface proteoglycans as a receptor.
First, they
demonstrated that AAV-2 binds to a cellufine sulfate column. Other viruses
known to
interact with such columns bind to negatively charged surface molecules (for
example,
several members of the Herpesviridae family known to use HS proteoglycans as
attachment receptors Compton et al., 1993; Mettenleiter et al., 1990; Sheik et
al.,
1992; WuDunn and Spear, 1989). Second, AAV can infect a wide variety of human,
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
19
rodent, and simian cell lines suggesting that it uses a ubiquitous cell
surface molecule
for infection (Muzyczka, 1992; Berns, 1996). One such family of ubiquitous
receptors is the proteoglycans family (Diertrich and Cassaro, 1977; Kjellen
and
Lindahl, 1991). Despite these correlations, Samulski and Summerford (1998)
concluded that although HS proteoglycans are required for AAV infection their
studies
did not address whether they are in fact sufficient. The present inventors
have shown
that while HSPG mediate the binding of AAV to the cell surface, they are
incapable of
mediating AAV entry into the cell. The present inventors, for the first time,
show that
efficient AAV mediated gene transfer requiremes FGFR as a co-receptor
C. Adeno-associated Virus and it use in Gene Therapy
Adeno-associated virus 2 (AAV)-based vectors have gained attention as a
useful alternative to the more commonly used retroviral and adenoviral vectors
for
human gene therapy. Although AAV utilizes the ubiquitously expressed cell
surface
heparan sulfate proteoglycan (HSPG) as a receptor, the transduction efficiency
of
AAV vectors varies greatly in different cells and tissues in vitro and in
vivo. The
present invention shows that cell surface expression of HSPG alone is
insufficient for
AAV infection, and that AAV also requires human fibroblast growth factor
receptor 1
(FGFRl) as a co-receptor for successful viral entry into the host cell. The
identification of FGFR 1 as a co-receptor for AAV provides new insights not
only into
its role in the life cycle of AAV, but also in the optimal use of AAV vectors
in human
gene therapy. The present section provides a discussion of the uses of AAV in
gene
therapy applications.
a. Adeno-associated Virus
AAV utilizes a linear, single-stranded DNA of about 4700 base pairs. Inverted
terminal repeats flank the genome. Two genes are present within the genome,
giving
rise to a number of distinct gene products. The first, the cap gene, produces
three
different virion proteins (VP), designated VP-l, VP-2 and VP-3. The second,
the rep
gene, encodes four non-structural proteins (NS). One or more of these rep gene
products is responsible for transactivating AAV transcription. The sequence of
AAV
is provided in (Srivastava et al., 1983).
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
The AAV-TTRs also contain an additional domain, designated the D-sequence,
a stretch of 20 nucleotides that is not involved in the HP formation (Berns
and
Bohenzky, 1987; Berns and Giraud, 1996; Srivastava et al., 1983), the
inventors
5 hypothesize that one or more cellular proteins) interact with the D-sequence
and
prevent the second strand viral DNA synthesis. Thus, the identification of
such a host
protein merits study. Once elucidated, it will be possible to increase the
transcription
and replication from an adeno-associated viral (AAV) vector. Other uses for
such a
protein will become apparent in the following disclosure.
The three promoters in AAV are designated by their location, in map units, in
the genome. These are, from left to right, p5, p 19 and p40. Transcription
gives rise to
six transcripts, two initiated at each of three promoters, with one of each
pair being
spliced. The splice site, derived from map units 42-46, is the same for each
transcript.
The four non-structural proteins apparently are derived from the longer of the
transcripts, and three virion proteins all arise from the smallest transcript.
AAV is not associated with any pathologic state in humans. Interestingly, for
efficient replication, AAV requires "helping" functions from viruses such as
herpes
simplex virus I and II, cytomegalovirus, pseudorabiesvirus and, of course,
adenovirus.
The best characterized of the helpers is adenovirus, and many "early"
functions for
this virus have been shown to assist with AAV replication. Low level
expression of
AAV rep proteins is believed to hold AAV structural expression in check, and
helper
virus infection is thought to remove this block.
The terminal repeats of the AAV vector of the present invention can be
obtained by restriction endonuclease digestion of AAV or a plasmid such as
p201,
which contains a modified AAV genome (Samulski et al., 1987), or by other
methods
known to the skilled artisan, including but not limited to chemical or
enzymatic
synthesis of the terminal repeats based upon the published sequence of AAV.
The
ordinarily skilled artisan can determine, by well-known methods such as
deletion
analysis, the minimum sequence or part of the AAV ITRs which is required to
allow
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
21
function, i.e. stable and site-specific integration. The ordinarily skilled
artisan also
can determine which minor modifications of the sequence can be tolerated while
maintaining the ability of the terminal repeats to direct stable, site-
specific integration.
b. Adeno-Associated Virus Mediated Gene Therapy
AAV-based vectors have proven to be safe and effective vehicle for gene
delivery in vitro, and these vectors are now being developed and tested in pre-
clinical
and clinical stages for a wide range of applications in potential gene
therapy, both ex
vivo and in vivo. However, the inventors (Ponnazhagan et al., 1997b; 1997c;
1997d;
1997d) and others (Carter and Flotte, 1996 ; Chatterjee et al., 1995; Ferrari
et al.,
1996; Fisher et al., 1996; Flotte et al., 1993; Goodman et al., 1994; Kaplitt
et al.,
1994; 1996, Kessler et al., 1996; Koeberl et al., 1997; Mizukami et al., 1996;
Xiao et
al., 1996) have repeatedly observed wide variations in AAV transduction
efficiency in
different cells and tissues in vitro as well as in vivo.
It would seem reasonable to suggest that AAV transduction efficiency
correlates with the number of the putative cell surface receptors, although
the identity
of this receptor still remains elusive (Mizukami et al., 1996). However, it
has become
clear from the inventors' present studies that such a correlation most
probably does not
exist since 293 cells that express relatively the least numbers of these
putative
receptors are transduced most efficiently, an observation consistent with
previously
published reports (Ferrari et al., 1996; Fisher et al., 1996).
AAV-mediated efficient gene transfer and expression in the lung already has
led to clinical trials for the treatment of cystic fibrosis (Carter and
Flotte, 1996; Flotte
et al., 1993). Similarly, the prospects for treatment of muscular dystrophy by
AAV-
mediated gene delivery of the dystrophin gene to skeletal muscle, of
Parkinson's
disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by
Factor
IX gene delivery to the liver, and potentially of myocardial infarction by
vascular
endothelial growth factor gene to the heart, appear promising since AAV-
mediated
transgene expression in these organs has recently been shown to be highly
efficient
(Fisher et al., 1996; Flotte et al., 1993; Kaplitt et al., 1994; 1996; Koeberl
et al., 1997;
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
22
McCown et al., 1996; Ping et al., 1996; Xiao et al., 1996). Since the present
invention shows that high efficiency of recombinant AAV transduction in these
organs or tissues requires the presence of an FGFR co-receptor along with the
presence of HSPG, any AAV-mediated gene therapy approach will be improved by
ensuring the presence of these receptors on the target cells. If such
receptors are not
endogenously expressed they can be engineered into the target cells/organs
thereby
ensuring an efficient binding and uptake of the AAV vector.
D. Additional Factors Involved in Efficient AAV-mediated Gene Transfer
Another aspect of the present invention involves increasing AAV-mediated
transgene expression by manipulating post-receptor entry cellular events. More
particularly, the examples of the present invention corroborate the inventors
earlier
findings that dephosphorylation of the ssD-BP is necessary to allow AAV-
mediated
transgene expression and AAV transduction efficiency.
Dephosphorylation of the D-BP facilitates second-strand synthesis of the AAV
genome delivered to target cells as a single-stranded DNA molecule, suggesting
that
manipulation of phosphorylation state of this protein may be exploitable as
one of the
strategies for significantly improving transduction efficiency of recombinant
AAV
vectors. A strong correlation between phosphorylation state of the D-BP and
the
extent of efficient transduction by AAV in murine organs/tissues in vivo has
also been
demonstrated, showing this approach of improving transduction efficiency will
work,
as well as indicating that the D-BP may be evolutionarily conserved.
The mechanism by which dephosphorylation of the D-BP facilitates second-
strand viral DNA synthesis remains unclear. One of the possibilities that the
D-BP
itself may possess a DNA polymerase-like activity currently is being tested.
Alternatively, dephosphorylation of the D-BP might activate cellular DNA
polymerase(s) necessary for host cell DNA synthesis or DNA-repair pathway, by
which the second-strand viral DNA synthesis is accomplished. The inventors'
studies
with highly purified preparations of the D-BP indicate that this protein
undergoes
auto-phosphorylation followed by auto-dephosphorylation, the significance of
which
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
23
is not clear. However, the purified D-BP has been determined to be an
approximately
53 kDa protein, but distinct from the p53 tumor suppressor protein, since
monoclonal
anti-p53 antibody failed to immunoprecipitate the D-BP.
Since the present invention shows that high efficiency of recombinant AAV
transduction in a variety of organs is most likely due to the presence of
dephosphorylated form of the D-BP, such an approach also will be useful in
determining the transduction potential of untested tissues/organs, especially
of human
origin, by AAV vectors. For example, based on the data shown in Table 4, it
would
appear that kidney might be an additional organ of choice for AAV-mediated
transduction since the ratio of dephosphorylated/phosphorylated D-BPs in these
tissues is approximately 1.4, a level consistent with that seen in 293 cells,
a cell line
derived from human embryonic kidney.
In another aspect, the search for additional specific compounds that mediate
dephosphorylation of the D-BP is facilitated by the present invention. The
elucidation
of such compounds will serve to augment transduction efficiency of recombinant
AAV vectors in a wide variety of tissue and organs, including primary
hematopoietic
stem/progenitor cells, potentially leading to their successful use in gene
therapy of
specific hematological disorders such as sickle-cell anemia and (3-thalassemia
(Goodman et al., 1994; Ponnazhagan et al., 1997d; Walsh et al., 1994; Zhou et
al.,
1996). Examples of mediators of phosphorylation known to those of skill in the
art
include genistein, tyrphostin A48, tyrphostin 1, tyrphostin 23, tyrphostin 25,
tyrphostin 46, tyrphostin 47, tyrphostin 51, tyrphostin 63, tyrphostin AG
1478,
herbmycin A, LY 294002, wortmannin, staurosporine, tyrphostin AG 126,
tyrphostin
AG 1288, tyrphostin 1295, and tyrphostin 1296. It is contemplated that the
tyrphostins group of inhibitors will be particularly useful in conjunction
with the
present invention.
Previously, the inventors further documented that treatment of cells with
specific inhibitors of the epidermal growth factor receptor protein tyrosine
kinase
(EGF-R PTK) activity, such as tyrphostin, leads to significant augmentation of
AAV
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
24
transduction efficiency, and phosphorylation of the ssD-BP is mediated by the
EGF-R
PTK (U.S. Serial Number 09/145,379, specifically incorporated herein by
reference
in its entirety). Treatment of cells with epidermal growth factor (EGF)
results in
phosphorylation of the ssD-BP, whereas treatment with tyrphostin causes
dephosphorylation of the ssD-BP, and consequently, leads to increased
expression of
the transgene. Furthermore, AAV transduction efficiency inversely correlates
with
expression of the EGF-R in different cell types, and stable transfection of
the EGF-R
cDNA causes phosphorylation of the ssD-BP leading to significant inhibition in
AAV-
mediated transgene expression which can be overcome by the tyrphostin
treatment.
E. Gene Transfer and Expression
a. Regulatory Elements
In describing both the AAV vector, which contains the transgene of interest,
and any FGFR or HSPG receptor containing construct for the purpose of
expressing a
protein, it should be noted that promoters will be required to drive the
transcription of
these genes. The nucleic acid encoding a gene product is under transcriptional
control
of a promoter. A "promoter" refers to a DNA sequence recognized by the
synthetic
machinery of the cell, or introduced synthetic machinery, required to initiate
the
specific transcription of a gene. The phrases "under transcriptional control"
or
"operably linked" mean that the promoter is in the correct location and
orientation in
relation to the nucleic acid to control RNA polymerise initiation and
expression of the
gene.
Within certain embodiments, expression vectors are employed to express the
receptor polypeptide for use in conjunction with AAV mediated gene therapy.
Expression requires that appropriate signals be provided in the vectors, and
which
include various regulatory elements, such as enhancers/promoters from both
viral and
mammalian sources that drive expression of the genes of interest in host
cells.
Elements designed to optimize messenger RNA stability and translatability in
host
cells also are defined. The conditions for the use of a number of dominant
drug
selection markers for establishing permanent, stable cell clones expressing
the
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
products also are provided, as is an element that links expression of the drug
selection
markers to expression of the polypeptide.
Throughout this application, the term "expression construct" is meant to
5 include any type of genetic construct containing a nucleic acid coding for a
gene
product in which part or all of the nucleic acid encoding sequence is capable
of being
transcribed. The transcript may be translated into a protein, but it need not
be. In
certain embodiments, expression includes both transcription of a gene and
translation
of mRNA into a gene product. In other embodiments, expression only includes
10 transcription of the nucleic acid encoding a gene of interest.
The term promoter will be used here to refer to a group of transcriptional
control modules that are clustered around the initiation site for RNA
polymerase II.
Much of the thinking about how promoters are organized derives from analyses
of
15 several viral promoters, including those for the HSV thymidine kinase (tk)
and SV40
early transcription units. These studies, augmented by more recent work, have
shown
that promoters are composed of discrete functional modules, each consisting of
approximately 7-20 by of DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
At least one module in each promoter functions to position the start site for
RNA synthesis. The best known example of this is the TATA box, but in some
promoters lacking a TATA box, such as the promoter for the mammalian terminal
deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a
discrete
element overlying the start site itself helps to fix the place of initiation.
Additional promoter elements regulate the frequency of transcriptional
initiation. Typically, these are located in the region 30-110 by upstream of
the start
site, although a number of promoters have recently been shown to contain
functional
elements downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is preserved when
elements
are inverted or moved relative to one another. In the tk promoter, the spacing
between
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
26
promoter elements can be increased to 50 by apart before activity begins to
decline.
Depending on the promoter, it appears that individual elements can function
either co-
operatively or independently to activate transcription.
The particular promoter employed to control the expression of a nucleic acid
sequence of interest is not believed to be important, so long as it is capable
of
direction the expression of the nucleic acid in the targeted cell. Thus, where
a human
cell is targeted, it is preferable to position the nucleic acid coding region
adjacent to
and under the control of a promoter that is capable of being expressed in a
human cell.
Generally speaking, such a promoter might include either a human or viral
promoter.
In various embodiments, the human cytomegalovirus (CMV) immediate early
gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal
repeat,
rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used
to
obtain high-level expression of the coding sequence of interest. The use of
other viral
or mammalian cellular or bacterial phage promoters which are well-known in the
art
to achieve expression of a coding sequence of interest is contemplated as
well,
provided that the levels of expression are sufficient for a given purpose.
By employing a promoter with well-known properties, the level and pattern of
expression of the protein of interest following transfection or transformation
can be
optimized. Further, selection of a promoter that is regulated in response to
specific
physiologic signals can permit inducible expression of the gene product.
Tables 2 and
3 list several elements/promoters which may be employed, in the context of the
present invention, to regulate the expression of the gene of interest. This
list is not
intended to be exhaustive of all the possible elements involved in the
promotion of
gene expression but, merely, to be exemplary thereof.
Enhancers are genetic elements that increase transcription from a promoter
located at a distant position on the same molecule of DNA. Enhancers are
organized
much like promoters. That is, they are composed of many individual elements,
each
of which binds to one or more transcriptional proteins.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
27
The basic distinction between enhancers and promoters is operational. An
enhancer region as a whole must be able to stimulate transcription at a
distance; this
need not be true of a promoter region or its component elements. On the other
hand, a
promoter must have one or more elements that direct initiation of RNA
synthesis at a
particular site and in a particular orientation, whereas enhancers lack these
specificities. Promoters and enhancers are often overlapping and contiguous,
often
seeming to have a very similar modular organization.
Below is a list of viral promoters, cellular promoters/enhancers and inducible
promoters/enhancers that could be used in combination with the nucleic acid
encoding
a gene of interest in an expression construct (Table 1 and Table 2).
Additionally, any
promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB)
could also be used to drive expression of the gene. Eukaryotic cells can
support
cytoplasmic transcription from certain bacterial promoters if the appropriate
bacterial
polymerise is provided, either as part of the delivery complex or as an
additional
genetic expression construct.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
28
TABLE 1
ENHANCER/PROMOTER
Immunoglobulin Heavy Chain
Immunoglobulin Light Chain
T-Cell Receptor
HLA DQ a and DQ (3
(3-Interferon
Interleukin-2
Interleukin-2 Receptor
MHC Class II 5
MHC Class II HLA-DRa
(3-Actin
Muscle Creatine Kinase
Prealbumin (Transthyretin)
Elastase 1
Metallothionein
Collagenase
Albumin Gene
a-Fetoprotein
i-Globin
(3-Globin
e-fos
c-HA-ras
Insulin
Neural Cell Adhesion Molecule (NCAM)
a 1-Antitrypsin
H2B (TH2B) Histone
Mouse or Type I Collagen
Glucose-Regulated Proteins (GRP94 and GRP78)
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
29
TABLE 1 (Continued)
Rat Growth Hormone
Human Serum Amyloid A (SAA)
i
Troponin I (TN n I
Platelet-Derived Growth Factor
Duchenne Muscular Dystrophy
S V40
Polyoma
Retroviruses
Papilloma Virus
Hepatitis B Virus
Human Immunodeficiency Virus
Cytomegalovirus
Gibbon Ape Leukemia Virus
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
TABLE 2
Element Inducer
MT II Phorbol Ester (TPA)
Heavy metals
MMTV (mouse mammary tumor Glucocorticoids
virus)
(3-Interferon poly(rT)X
poly(rc)
Adenovirus 5 E2 Ela
c-jun Phorbol Ester (TPA), H202
Collagenase Phorbol Ester (TPA)
Stromelysin Phorbol Ester (TPA), IL,-1
SV40 Phorbol Ester (TPA)
Murine MX Gene Interferon, Newcastle
Disease
Virus
GRP78 Gene A23187
a-2-Macroglobulin IL-6
Vimentin Serum
MHC Class I Gene H-2kB Interferon
HSP70 Ela, S V40 Large T Antigen
Proliferin Phorbol Ester-TPA
Tumor Necrosis Factor FMA
Thyroid Stimulating HormoneThyroid Hormone
a Gene
Insulin E Box Glucose
Where a cDNA insert is employed, one typically will desire to include a
5 polyadenylation signal to effect proper polyadenylation of the gene
transcript. The
nature of the polyadenylation signal is not believed to be crucial to the
successful
practice of the invention, and any such sequence may be employed such as human
growth hormone and SV40 polyadenylation signals. Also contemplated as an
element
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
31
of the expression cassette is a terminator. These elements can serve to
enhance
message levels and to minimize read through from the cassette into other
sequences.
b. Transgene Constructs
Transgene expression will be driven by a selected promoter. The promoter
selection will depend on the polypeptide to be expressed, the target tissue
and the
purpose for expression. For example, if the protein is simply to be produced
in vitro
and purified, a high level promoter will be utilized. If the protein is toxic
to the cells,
it may be desirable to regulate the expression of the protein such that cells
proliferation is maximized prior to polypeptide expression. If the protein's
processing
or secretion is dependent upon a particular stage in the host cell's cycle, it
may be
desirable to employ a promoter that is regulated in an appropriate, cell cycle
dependentfashion.
c. Selectable Markers
In certain embodiments of the invention, the cells contain nucleic acid
constructs of the present invention, a cell may be identified in vitro or in
vivo by
including a marker in the expression construct. Such markers would confer an
identifiable change to the cell permitting easy identification of cells
containing the
expression construct. Usually the inclusion of a drug selection marker aids in
cloning
and in the selection of transformants, for example, genes that confer
resistance to
neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful
selectable markers. Alternatively, enzymes such as herpes simplex virus
thymidine
kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
Immunologic markers also can be employed. The selectable marker employed is
not
believed to be important, so long as it is capable of being expressed
simultaneously
with the nucleic acid encoding a gene product. Further examples of selectable
markers are well known to one of skill in the art.
d. Multigene Constructs and IRES
In certain embodiments of the invention, the use of internal ribosome binding
sites (IRES) elements are used to create multigene, or polycistronic,
messages. IRES
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
32
elements are able to bypass the ribosome scanning model of 5' methylated Cap
dependent translation and begin translation at internal sites (Pelletier and
Sonenberg,
1988). IRES elements from two members of the picanovirus family (polio and
encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as
well
an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements
can be linked to heterologous open reading frames. Multiple open reading
frames can
- be transcribed together, each separated by an IRES, creating polycistronic
messages.
By virtue of the IRES element, each open reading frame is accessible to
ribosomes for
efficient translation. Multiple genes can be efficiently expressed using a
single
promoter/enhancer to transcribe a single message.
Any heterologous open reading frame can be linked to IRES elements. This
includes genes for secreted proteins, mufti-subunit proteins, encoded by
independent
genes, intracellular or membrane-bound proteins and selectable markers. In
this way,
expression of several proteins can be simultaneously engineered into a cell
with a
single construct and a single selectable marker.
e. Delivery of Expression Vectors
The present application proposes the use of AAV expression vectors for
delivering a gene to a particular host or target cell. However, as noted
above, such
host cells also require the presence of an FGFR and an HSPG receptor for
efficient
AAV infection so that the transgene may be efficiently taken up and expressed.
Thus,
in order to increase the efficiency of AAV-mediated gene delivery, it will be
desirable
to stimulate, increase or introduce an FGFR and HSPG receptor activity of the
host
cell. This may be achieved by delivering a gene encoding the receptor to the
target
cell. This delivery may be achieved using viral or non-viral delivery vectors.
In one embodiment, it may be useful to employ viruses other than AAV to
deliver the FGFR and HSPG receptor expression construct. Such viruses may
include
those that enter cells via receptor-mediated endocytosis, integrate into host
cell
genome and express viral genes stably and efficiently (Ridgeway, 1988; Nicolas
and
Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). These DNA viruses
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
33
including the papovaviruses (simian virus 40, bovine papilloma virus, and
polyoma)
(Ridgeway, 1988; Baichwal and Sugden, 1986), adenoviruses (Ridgeway, 1988;
Baichwal and Sugden, 1986) and retroviruses (Coffin, 1990; Mann et al., 1983;
Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983).
In other embodiments, non-viral transfer is contemplated. Several non-viral
methods for the transfer of HSPG and/or FGFR receptor expression constructs
into
cultured mammalian cells are contemplated by the present invention. These
include
calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and
Okayama,
1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-
Kaspa et
al., 1986; Potter et al., 1984), direct microinjection (Harland and Weintraub,
1985),
DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al., 1979) and
lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene
bombardment using high velocity microprojectiles (Yang et al., 1990), and
receptor-
mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988). Some of these
techniques may be successfully adapted for in vivo or ex vivo use.
Once the expression construct has been delivered into the cell, the nucleic
acid
encoding the gene of interest may be positioned and expressed at different
sites. In
certain embodiments, the nucleic acid encoding the gene may be stably
integrated into
the genome of the cell. This integration may be in the cognate location and
orientation via homologous recombination (gene replacement) or it may be
integrated
in a random, non-specific location (gene augmentation). In yet further
embodiments,
the nucleic acid may be stably maintained in the cell as a separate, episomal
segment
of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient
to
permit maintenance and replication independent of or in synchronization with
the host
cell cycle. How the expression construct is delivered to a cell and where in
the cell
the nucleic acid remains is dependent on the type of expression construct
employed.
In yet another embodiment of the invention, the expression construct
containing the receptor gene may simply consist of naked recombinant DNA or
plasmids. Transfer of the construct may be performed by any of the methods
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
34
mentioned above which physically or chemically permeabilize the cell membrane.
This is particularly applicable for transfer in vitro but it may be applied to
in vivo use
as well. Dubensky et al. (1984) successfully injected polyomavirus DNA in the
form
of calcium phosphate precipitates into liver and spleen of adult and newborn
mice
demonstrating active viral replication and acute infection. Benvenisty and
Neshif
(1986) also demonstrated that direct intraperitoneal injection of calcium
phosphate-
precipitated plasmids results in expression of the transfected genes. It is
envisioned
that DNA encoding a gene of interest may also be transferred in a similar
manner in
vivo and express the gene product.
In still another embodiment of the invention for transferring a naked DNA
expression construct into cells may involve particle bombardment. This method
depends on the ability to accelerate DNA-coated microprojectiles to a high
velocity
allowing them to pierce cell membranes and enter cells without killing them
(Klein et
al., 1987). Several devices for accelerating small particles have been
developed. One
such device relies on a high voltage discharge to generate an electrical
current, which
in turn provides the motive force (Yang et al., 1990). The microprojectiles
used have
consisted of biologically inert substances such as tungsten or gold beads.
Selected organs including the liver, skin, and muscle tissue of rats and mice
have been bombarded in vivo (Yang et al., 1990; Zelenin et al., 1991 ). This
may
require surgical exposure of the tissue or cells, to eliminate any intervening
tissue
between the gun and the target organ, i. e. ex vivo treatment. Again, DNA
encoding a
particular gene may be delivered via this method and still be incorporated by
the
present invention.
In a further embodiment of the invention, the expression construct containing
may be entrapped in a liposome. Liposomes are vesicular structures
characterized by
a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar
liposomes have multiple lipid layers separated by aqueous medium. They form
spontaneously when phospholipids are suspended in an excess of aqueous
solution.
The lipid components undergo self-rearrangement before the formation of closed
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
structures and entrap water and dissolved solutes between the lipid bilayers
(Ghosh
and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
Liposome-mediated nucleic acid delivery and expression of foreign DNA in
5 vitro has been very successful. Wong et al., (1980) demonstrated the
feasibility of
liposome-mediated delivery and expression of foreign DNA in cultured chick
embryo,
HeLa and hepatoma cells. Nicolau et al., ( 1987) accomplished successful
liposome-
mediated gene transfer in rats after intravenous injection.
10 In certain embodiments of the invention, the liposome may be complexed with
a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with
the cell
membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al.,
1989). In other embodiments, the liposome may be complexed or employed in
conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et
al.,
15 1991 ). In yet further embodiments, the liposome may be complexed or
employed in
conjunction with both HVJ and HMG-1. In that such expression constructs have
been
successfully employed in transfer and expression of nucleic acid in vitro and
irc vivo,
then they are applicable for the present invention. Where a bacterial promoter
is
employed in the DNA construct, it also will be desirable to include within the
20 liposome an appropriate bacterial polymerise.
Other expression constructs which can be employed to deliver a nucleic acid
encoding a particular gene into cells are receptor-mediated delivery vehicles.
These
take advantage of the selective uptake of macromolecules by receptor-mediated
25 endocytosis in almost all eukaryotic cells. Because of the cell type-
specific
distribution of various receptors, the delivery can be highly specific (Wu and
Wu,
1993).
Receptor-mediated gene targeting vehicles generally consist of two
30 components: a cell receptor-specific ligand and a DNA-binding agent.
Several
ligands have been used for receptor-mediated gene transfer. The most
extensively
characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
36
transferrin (Wagner et al., 1990). Recently, a synthetic neoglycoprotein,
which
recognizes the same receptor as ASOR, has been used as a gene delivery vehicle
(Ferkol et al., 1993; Perales et al., 1994) and epidermal growth factor (EGF)
has also
been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).
In other embodiments, the delivery vehicle may comprise a ligand and a
liposome. For example, Nicolau et al., ( 1987) employed lactosyl-ceramide, a
galactose-terminal asialganglioside, incorporated into liposomes and observed
an
increase in the uptake of the insulin gene by hepatocytes. Thus, it is
feasible that a
nucleic acid encoding a particular gene also may be specifically delivered
into a cell
type such as lung, epithelial or tumor cells, by any number of receptor-ligand
systems
with or without liposomes. For example, epidermal growth factor (EGF) may be
used
as the receptor for mediated delivery of a nucleic acid encoding a gene in
many tumor
cells that exhibit upregulation of EGF receptor. Mannose can be used to target
the
mannose receptor on liver cells. Also, antibodies to CDS (CLL), CD22
(lymphoma),
CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting
moieties.
In certain embodiments, gene transfer may more easily be performed under ex
vivo conditions. Ex vivo gene therapy refers to the isolation of cells from an
animal,
the delivery of a nucleic acid into the cells in vitro, and then the return of
the modified
cells back into an animal. This may involve the surgical removal of
tissue/organs
from an animal or the primary culture of cells and tissues.
F. Propagation of AAV Vectors and Transformation of Host Cells
The following is an exemplary description of the propagation of the AAV
vectors of the present invention, of course the conditions described are only
exemplary and in light of the present disclosure it will be possible for one
of ordinary
skill in the art to modify these propagation conditions according to
particular needs.
Transfer of the plasmid may be accomplished any standard gene transfer
mechanism:
calcium phosphate precipitation, lipofection, electroporation, microprojectile
bombardment or other suitable means. Following transfer, host cells may
further be
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
37
infected with a helper virus and the virions are isolated and helper virus is
inactivated
(e.g., heated at 56°C for one h). The resulting helper free stocks of
virions are used to
infect appropriate target cells. Mature virions may further be isolated by
standard
methods, e.g., cesium chloride centrifugation, and to inactivate any
contaminating
adenovirus.
Function of the vectors of the present invention, i.e. the ability to mediate
transfer and expression of the heterologous gene in hematopoietic stem or
progenitor
cells, can be evaluated by monitoring the expression of the heterologous gene
in
transduced cells. Obviously, the assay for expression depends upon the nature
of the
heterologous gene. Expression can be monitored by a variety of methods
including
immunological, histochemical or activity assays. For example, Northern
analysis can
be used to assess transcription using appropriate DNA or RNA probes. If
antibodies
to the polypeptide encoded by the heterologous gene are available, Western
blot
analysis, immunohistochemistry or other immunological techniques can be used
to
assess the production of the polypeptide. Appropriate biochemical assays also
can be
used if the heterologous gene is an enzyme. For example, if the heterologous
gene
encodes antibiotic resistance, a determination of the resistance of infected
cells to the
antibiotic can be used to evaluate expression of the antibiotic resistance
gene.
Site-specific integration can be assessed, for example, by Southern blot
analysis. DNA is isolated from cells transduced by the vectors of the present
invention, digested with a variety of restriction enzymes, and analyzed on
Southern
blots with an AAV-specific probe. A single band of hybridization evidences
site-
specific integration. Other methods known to the skilled artisan, such as
polymerase
chain reaction (PCR) analysis of chromosomal DNA can be used to assess stable
integration.
G. Cell Culture and Selection
In one embodiment, the present invention contemplates the use of AAV vectors
to transform cells for the production of mammalian cell cultures for use in
the various
therapeutic aspects of the present invention. In order for the cells to be
kept viable while
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
38
in vitro and in contact with the expression construct, it is necessary to
ensure that the
cells are maintained with the correct ratio of oxygen and carbon dioxide and
nutrients,
but are protected from microbial contamination. Cell culture techniques are
well
documented and are disclosed herein by reference (Freshner, 1992).
The construct encoding the protein of interest may be transferred by the viral
vector, as described above, into appropriate host cells followed by culture of
cells
under the appropriate conditions. The gene for virtually any polypeptide may
be
employed in this manner. Examples of useful mammalian cell lines are those
that
express the appropriate receptor for B 19 virus. These include cells derived
from bone
marrow cells, peripheral blood cells and fetal liver cells.
Bone marrow cells are isolated and enriched for hematopoietic stem cells
(HSC), e.g., by fluorescence activated cell sorting as described in Srivastava
et al.
(1988). HSC are capable of self-renewal as well as initiating long-term
hematopoiesis
and differentiation into multiple hematopoietic lineages in vitro. HSC are
transfected
with the vector of the present invention or infected with varying
concentrations of
virions containing a subject hybrid vector and then assessed for the
expression of the
heterologous gene. The assay for expression depends upon the nature of the
heterologous gene. Expression can be monitored by a variety of methods
including
immunological, histochemical or activity assays. For example, Northern
analysis can
be used to assess transcription using appropriate DNA or RNA probes. If
antibodies to
the polypeptide encoded by the heterologous gene are available, Western blot
analysis,
immunohistochemistry or other immunological techniques can be used to assess
the
production of the polypeptide. Appropriate biochemical assays also can be used
if the
heterologous gene is an enzyme. For example, if the heterologous gene encodes
antibiotic resistance, a determination of the resistance of infected cells to
the antibiotic
can be used to evaluate expression of the antibiotic resistance gene.
An important consideration is the appropriate modification needed for a
particular polypeptide. Such modifications (e.g., glycosylation) and
processing (e.g.,
cleavage) of protein products may be important for the function of the
protein.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
39
Different host cells have characteristic and specific mechanisms for the post-
translational processing and modification of proteins. Appropriate cell lines
or host
systems can be chosen to insure the correct modification and processing of the
protein
expressed.
Examples of useful mammalian host cell lines are Vero and HeLa cells and
cell lines of Chinese hamster ovary, W 138, BHK, COS-7, 293, HepG2, NIH3T3,
RIN
and MDCK cells. In addition, a host cell strain may be chosen that modulates
the
expression of the inserted sequences, or modifies and process the gene product
in the
manner desired. Such modifications (e.g., glycosylation) and processing (e.g.,
cleavage) of protein products may be important for the function of the
protein.
Different host cells have characteristic and specific mechanisms for the post-
translational processing and modification of proteins. Appropriate cell lines
or host
systems can be chosen to insure the correct modification and processing of the
foreign
protein expressed.
A number of selection systems may be used including, but not limited to, HSV
thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase and adenine
phosphoribosyltransferase genes, in tk-, hgprt- or aprt- cells, respectively.
Also, anti-
metabolite resistance can be used as the basis of selection for dhfr, that
confers
resistance to; gpt, that confers resistance to mycophenolic acid; neo, that
confers
resistance to the aminoglycoside 6418; and hygro, that confers resistance to
hygromycin.
Animal cells can be propagated in vitro in two modes: as non-anchorage
dependent cells growing in suspension throughout the bulk of the culture or as
anchorage-dependent cells requiring attachment to a solid substrate for their
propagation (i.e. a monolayer type of cell growth).
Non-anchorage dependent or suspension cultures from continuous established
cell lines are the most widely used means of large scale production of cells
and cell
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
products. However, suspension cultured cells have limitations, such as
tumorigenic
potential and lower protein production than adherent T-cells.
Large scale suspension culture of mammalian cells in stirred tanks is a
5 common method for production of recombinant proteins. Two suspension culture
reactor designs are in wide use - the stirred reactor and the airlift reactor.
The stirred
design has successfully been used on an 8000 liter capacity for the production
of
interferon. Cells are grown in a stainless steel tank with a height-to-
diameter ratio of
1:1 to 3:1. The culture is usually mixed with one or more agitators, based on
bladed
10 disks or marine propeller patterns. Agitator systems offering less shear
forces than
blades have been described. Agitation may be driven either directly or
indirectly by
magnetically coupled drives. Indirect drives reduce the risk of microbial
contamination through seals on stirrer shafts.
15 The airlift reactor, also initially described for microbial fermentation
and later
adapted for mammalian culture, relies on a gas stream to both mix and
oxygenate the
culture. The gas stream enters a riser section of the reactor and drives
circulation.
Gas disengages at the culture surface, causing denser liquid free of gas
bubbles to
travel downward in the downcomer section of the reactor. The main advantage of
this
20 design is the simplicity and lack of need for mechanical mixing. Typically,
the
height-to-diameter ratio is 10:1. The airlift reactor scales up relatively
easily, has
good mass transfer of gases and generates relatively low shear forces.
The antibodies of the present invention are particularly useful for the
isolation
25 of antigens by immunoprecipitation. Immunoprecipitation involves the
separation of
the target antigen component from a complex mixture, and is used to
discriminate or
isolate minute amounts of protein. For the isolation of membrane proteins
cells must
be solubilized into detergent micelles. Nonionic salts are preferred, since
other agents
such as bile salts, precipitate at acid pH or in the presence of bivalent
canons.
30 Antibodies are and their uses are discussed further, below.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
41
H. Transgenes
For gene therapy with an AAV vector, virtually any transgene may be utilized
in the vectors described herein. In a preferred embodiment, the heterologous
gene
encodes a biologically functional protein, i.e. a polypeptide or protein which
affects
the cellular mechanism of a cell in which the biologically functional protein
is
expressed. For example, the biologically functional protein can be a protein
which is
essential for normal growth of the cell or for maintaining the health of a
mammal.
The biologically functional protein also can be a protein which improves the
health of
a mammal by either supplying a missing protein, by providing increased
quantities of
a protein which is underproduced in the mammal or by providing a protein which
inhibits or counteracts an undesired molecule which may be present in the
mammal.
The biologically functional protein also can be a protein which is a useful
protein for
investigative studies for developing new gene therapies or for studying
cellular
mechanisms.
Expression of several proteins that are normally secreted can be engineered
into cells. The cDNA's encoding a number of useful human proteins are
available.
Examples would include soluble CD-4, Factor VIII, Factor IX, von Willebrand
Factor,
TPA, urokinase, hirudin, interferons, TNF, interleukins, hematopoietic growth
factors,
antibodies, albumin, leptin, transferin and nerve growth factors.
Expression of non-secreted proteins can be engineered into cells. Two general
classes of such proteins can be defined. The first are proteins that, once
expressed in
cells, stay associated with the cells in a variety of destinations. These
destinations
include the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, Golgi,
membrane of secretory granules and plasma membrane. Non-secreted proteins are
both soluble and membrane associated. The second class of proteins are ones
that are
normally associated with the cell, but have been modified such that they are
now
secreted by the cell. Modifications would include site-directed mutagenesis or
expression of truncations of engineered proteins resulting in their secretion
as well as
creating novel fusion proteins that result in secretion of a normally non-
secreted
protein.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
42
p53 currently is recognized as a tumor suppressor gene (Montenarh, 1992).
High levels of mutant p53 have been found in many cells transformed by
chemical
carcinogenesis, ultraviolet radiation, and several viruses, including SV40.
The p53
gene is a frequent target of mutational inactivation in a wide variety of
human tumors
and is already documented to be the most frequently-mutated gene in common
human
cancers (Mercer, 1992). It is mutated in over 50% of human NSCLC (Hollestein
et
al., 1991) and in a wide spectrum of other tumors.
The p53 gene encodes a 393-amino-acid phosphoprotein that can form
complexes with host proteins such as large-T antigen and E1B. The protein is
found
in normal tissues and cells, but at concentrations which are generally minute
by
comparison with transformed cells or tumor tissue. Interestingly, wild-type
p53
appears to be important in regulating cell growth and division. Overexpression
of
wild-type p53 has been shown in some cases to be anti-proliferative in human
tumor
cell lines. Thus, p53 can act as a negative regulator of cell growth
(Weinberg, 1991 )
and may directly suppress uncontrolled cell growth or directly or indirectly
activate
genes that suppress this growth. Thus, absence or inactivation of wild-type
p53 may
contribute to transformation. However, some studies indicate that the presence
of
mutant p53 may be necessary for full expression of the transforming potential
of the
gene.
Wild-type p53 is recognized as an important growth regulator in many cell
types. Missense mutations are common for the p53 gene and are known to occur
in at
least 30 distinct codons, often creating dominant alleles that produce shifts
in cell
phenotype without a reduction to homozygosity. Additionally, many of these
dominant negative alleles appear to be tolerated in the organism and passed on
in the
germ line. Various mutant alleles appear to range from minimally dysfunctional
to
strongly penetrant, dominant negative alleles (Weinberg, 1991 ).
Casey and colleagues have reported that transfection of DNA encoding wild-
type p53 into two human breast cancer cell lines restores growth suppression
control
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
43
in such cells (Casey et al., 1991). A similar effect has also been
demonstrated on
transfection of wild-type, but not mutant, p53 into human lung cancer cell
lines
(Takahasi et al., 1992). p53 appears dominant over the mutant gene and will
select
against proliferation when transfected into cells with the mutant gene. Normal
expression of the transfected p53 is not detrimental to normal cells with
endogenous
wild-type p53. Thus, such constructs might be taken up by normal cells without
adverse effects. It is thus proposed that the treatment of p53-associated
cancers with
wild-type p53 expression constructs will reduce the number of malignant cells
or their
growth rate. Furthermore, recent studies suggest that some p53 wild-type
tumors are
also sensitive to the effects of exogenous p53 expression.
The major transitions of the eukaryotic cell cycle are triggered by cyclin-
dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4 (CDK4),
regulates progression through the G1 phase. The activity of this enzyme may be
to
phosphorylate Rb at late G~. The activity of CDK4 is controlled by an
activating
subunit, D-type cyclin, and by an inhibitory subunit, e.g., pl6INKa ,which has
been
biochemically characterized as a protein that specifically binds to and
inhibits CDK4,
and thus may regulate Rb phosphorylation (Serrano et al., 1993; Serrano et
al., 1995).
Since the pl6INxa protein is a CDK4 inhibitor (Serrano, 1993), deletion of
this gene
may increase the activity of CDK4, resulting in hyperphosphorylation of the Rb
protein. p 16 also is known to regulate the function of CDK6.
pl6INK4 belongs to a newly described class of CDK-inhibitory proteins that
also includes pl6B, p2lWAFi, cm, sDn and p27K~1. The p16INK4 gene maps to
9p21, a
chromosome region frequently deleted in many tumor types. Homozygous deletions
and mutations of the p16INK4 gene are frequent in human tumor cell lines. This
evidence suggests that the p16~K4 gene is a tumor suppressor gene. This
interpretation has been challenged, however, by the observation that the
frequency of
the pl6INKa gene alterations is much lower in primary uncultured tumors than
in
cultured cell lines (Caldas et al., 1994; Cheng et al., 1994; Hussussian et
al., 1994;
Kamb et al., 1994a; Kamb et al., 1994b; Mori et al., 1994; Okamoto et al.,
1994;
Nobori et al., 1995; Orlow et al., 1994; Arap et al., 1995). Restoration of
wild-type
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
44
p16INK4 function by transfection with a plasmid expression vector reduced
colony
formation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).
C-CAM is expressed in virtually all epithelial cells (Odin and Obrink, 1987).
C-CAM, with an apparent molecular weight of 105 kD, was originally isolated
from
the plasma membrane of the rat hepatocyte by its reaction with specific
antibodies that
neutralize cell aggregation (Obrink, 1991). Recent studies indicate that,
structurally,
C-CAM belongs to the immunoglobulin (Ig) superfamily and its sequence is
highly
homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti, 1989). Using a
baculovirus expression system, Cheung et al. (1993a; 1993b and 1993c)
demonstrated
that the first Ig domain of C-CAM is critical for cell adhesion activity.
Cell adhesion molecules, or CAMs are known to be involved in a complex
network of molecular interactions that regulate organ development and cell
differentiation (Edelman, 1985). Recent data indicate that aberrant expression
of
CAMs may be involved in the tumorigenesis of several neoplasms; for example,
decreased expression of E-cadherin, which is predominantly expressed in
epithelial
cells, is associated with the progression of several kinds of neoplasms
(Edelman and
Crossin, 1991; Frixen et al., 1991; Bussemakers et al., 1992; Matsura et al.,
1992;
Umbas et al., 1992). Also, Giancotti and Ruoslahti (1990) demonstrated that
increasing expression of a.s(31 integrin by gene transfer can reduce
tumorigenicity of
Chinese hamster ovary cells in vivo. C-CAM now has been shown to suppress
tumor
growth in vitro and in vivo.
Other tumor suppressors that may be employed according to the present
invention include RB, APC, DCC, NF-l, NF-2, WT-1, MEN-I, MEN-II, zacl, p73,
BRCA1, VHL, FCC, MMAC1, MCC, p16, p21, p57, C-CAM, p27 and BRCA2.
Inducers of apoptosis, such as Bax, Bak, Bcl-XS, Bik, Bid, Harakiri, Ad E1B,
Bad and
ICE-CED3 proteases, similarly could find use according to the present
invention.
Various enzyme genes are of interest according to the present invention. Such
enzymes include cytosine deaminase, hypoxanthine-guanine
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
phosphoribosyltransferase, galactose-1-phosphate uridyltransferase,
phenylalanine
hydroxylase, glucocerbrosidase, sphingomyelinase, a-L-iduronidase, glucose-6-
phosphate dehydrogenase, HSV thymidine kinase and human thymidine kinase.
5 Hormones are another group of gene that may be used in the vectors described
herein. Included are growth hormone, prolactin, placental lactogen,
luteinizing
hormone, follicle-stimulating hormone, chorionic gonadotropin, thyroid-
stimulating
hormone, leptin, adrenocorticotropin (ACTH), angiotensin I and II, (3-
endorphin, (3-
melanocyte stimulating hormone ((3-MSH), cholecystokinin, endothelin I,
galanin,
10 gastric inhibitory peptide (GIP), glucagon, insulin, lipotropins,
neurophysins,
somatostatin, calcitonin, calcitonin gene related peptide (CGRP), (3-
calcitonin gene
related peptide, hypercalcemia of malignancy factor (1-40), parathyroid
hormone-
related protein (107-139) (PTH-rP), parathyroid hormone-related protein (107-
111)
(PTH-rP), glucagon-like peptide (GLP-1), pancreastatin, pancreatic peptide,
peptide
15 YY, PHM, secretin, vasoactive intestinal peptide (VIP), oxytocin,
vasopressin (AVP),
vasotocin, enkephalinamide, metorphinamide, alpha melanocyte stimulating
hormone
(alpha-MSH), atrial natriuretic factor (5-28) (ANF), amylin, amyloid P
component
(SAP-1), corticotropin releasing hormone (CRH), growth hormone releasing
factor
(GHRH), luteinizing hormone-releasing hormone (LHRH), neuropeptide Y,
20 substance K (neurokinin A ), substance P and thyrotropin releasing hormone
(TRH).
The cDNA's encoding a number of therapeutically useful human proteins are
available. Other proteins include protein processing enzymes such as PC2 and
PC3,
and PAM, transcription factors such as IPF 1, and metabolic enzymes such as
adenosine deaminase, phenylalanine hydroxylase, glucocerebrosidase.
Other classes of genes that are contemplated to be inserted into the vectors
of
the present invention include interleukins and cytokines. Interleukin 1 (IL-
1), IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL,-10, IL-11 IL-12, GM-CSF and G-
CSF.
Examples of diseases for which the present viral vector would be useful
include, but are not limited to, adenosine deaminase deficiency, human blood
clotting
factor IX deficiency in hemophilia B, and cystic fibrosis, which would involve
the
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
46
replacement of the cystic fibrosis transmembrane receptor gene. The vectors
embodied in the present invention could also be used for treatment of
hyperproliferative disorders such as rheumatoid arthritis or restenosis by
transfer of
genes encoding angiogenesis inhibitors or cell cycle inhibitors. Transfer of
prodrug
activators such as the HSV-TK gene can be also be used in the treatment of
hyperproliferative disorders, including cancer.
Other therapeutics genes might include genes encoding antigens such as viral
antigens, bacterial antigens, fungal antigens or parasitic antigens. Viruses
include
picornavirus, coronavirus, togavirus, flavirviru, rhabdovirus, paramyxovirus,
orthomyxovirus, bunyavirus, arenvirus, reovirus, retrovirus, papovavirus,
parvovirus,
herpesvirus, poxvirus, hepadnavirus, and spongiform virus. Preferred viral
targets
include influenza, herpes simplex virus 1 and 2, measles, small pox, polio or
HIV.
Pathogens include trypanosomes, tapeworms, roundworms, helminths, . Also,
tumor
markers, such as fetal antigen or prostate specific antigen, may be targeted
in this
manner. Preferred examples include HIV env proteins and hepatitis B surface
antigen.
Administration of a vector according to the present invention for vaccination
purposes
would require that the vector-associated antigens be sufficiently non-
immunogenic to
enable long term expression of the transgene, for which a strong immune
response
would be desired. Preferably, vaccination of an individual would only be
required
infrequently, such as yearly or biennially, and provide long term immunologic
protection against the infectious agent.
Cells engineered to produce such proteins could be used for either in vitro
production of the protein or for in vivo, cell-based therapies. In vitro
production
would entail purification of the expressed protein from either the cell pellet
for
proteins remaining associated with the cell or from the conditioned media from
cells
secreting the engineered protein. In vivo, cell-based therapies would either
be based
on secretion of the engineered protein or beneficial effects of the cells
expressing a
non-secreted protein.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
47
Engineering mutated, truncated or fusion proteins into cells also is
contemplated. Examples of each type of engineering resulting in secretion of a
protein are given (Ferber et al., 1991; Mains et al., 1995). Reviews on the
use of such
proteins for studying the regulated secretion pathway also are cited (Burgess
and
Kelly, 1987; Chavez et al., 1994).
I. Methods of Altering Receptor Activity
As stated above, the present invention provides methods for increasing the
transduction efficiency of AAV infection in, for example, gene therapy. These
methods exploit the inventors' observation, described in detail herein, that
expression
of the FGFR receptor is required, in addition to cell surface expression of
HSPG, for
efficient AAV infection. It is, therefore, a goal of the present invention to
exploit this
observation. This exploitation takes, basically, two forms. First, by
observing the
FGFR receptor expression of a cell, alone or in conjunction with the
observation of
HSPG expression, one can determine the susceptibility of that cell to
infection by
AAV (see U.S. Patents 5,229,501 and 5,707,632). Second, upon a determination
that
one or both of these receptors are missing from a target cell, it is possible
to render
that target cell susceptible by increasing the presence of one or both of
these receptors
on the cell surface.
There are at least three different methods through which FGFR receptor
expression on the cell surface may be increased. First, one may simply
increase the
amount of mature endogenous FGFR receptor that is contained in the cell, with
the
expected result of increased cell surface expression. This may involve
increasing
transcription, translation, or post-translational processing. It also may
involve
increasing the stability of FGFR, for example, by reducing turnover or
receptor
cycling. While the regulation of FGFR is not completely elucidated, many cell
surface receptor molecules exhibit a complex, autoregulatory mechanism,
whereby
binding and internalization of the receptor-ligand complex stimulates
increased
transcription and translation of the receptor. Therefore, a fruitful approach
may
involve the use of FGFR peptides or analogs that, following internalization,
cause
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
48
increased expression of the receptor. Classic pharmaceuticals also may be
utilized
which affect FGFR levels.
Second, one may provide a cell with an FGFR gene, in an expression
construct, that will facilitate expression of the receptor in increased
quantities. The
various aspects of this embodiment are described in detail throughout the
relevant
portions of this document. Briefly, one would provide a target cells with an
appropriate vector encoding the FGFR gene of choice, under the control of a
promoter
active in that target cell. The promoter, either constitutively or under some
form of
induction, would stimulate transcription of the FGFR gene, resulting in
expression of
the receptor. Subsequent infection of the cell with an AAV particle should be
enhanced by the increased cell surface FGFR.
Third, one may directly increase the ability of existing FGFR receptor to bind
AAV by treating the cell with an agent, presumably one that binds to the FGFR
protein or a closely related molecules. Many receptors exhibit so-called
"allosteric"
effects, where binding of a ligand to one part of the molecule will affect the
structure,
and possibly function, of another part. In this embodiment, FGF peptides may
serve
themselves, or as useful models for other molecules, which bind FGFR and
improve
the receptor's ability to bind AAV.
J. Methods of Therapy
The vectors of the present invention are useful for gene therapy, the therapy
consists of administering vector and increasing, stimulating or otherwise
providing a
function FGFR or HSPG receptor activity/function to the host cell. In
particular
embodiments, the vectors of the present invention can direct cell-specific
expression
of a desired gene, and thus are useful in the treatment of hemoglobinopathies.
Such
maladies include thalassemia, sickle-cell anemia, diabetes, and cancer. The
heterologous gene, in this context, can be the normal counterpart of one that
is
abnormally produced or underproduced in the disease state, for example (3-
globin for
the treatment of sickle-cell anemia, and a-globin, (3-globin or y-globin in
the treatment
of thalassemia. The heterologous gene can encode antisense RNA as described
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
49
hereinabove. For example, a-globin is produced in excess over (3-globin in (3-
thalassemia. Accordingly, (3-thalassemia can be treated in accordance with the
present
invention by gene therapy with a vector in which the heterologous gene encodes
an
antisense RNA. The antisense RNA is selected such that it binds to a target
sequence
of the a-globin mRNA to prevent translation of a-globin, or to a target
sequence of
the a-globin DNA such that binding prevents transcription of a-globin DNA. In
the
treatment of cancer the heterologous gene can be a gene associated with tumor
suppression, such as retinoblastoma gene, p53, p16, p21 or the gene encoding
tumor
necrosis factor.
The use of the vectors of the present invention for the treatment of disease
involves, in one embodiment, the transduction of hematopoeitic stems cells or
progenitor cells with the claimed vectors in combination with the provision of
an
active receptor that will mediate the efficient infection of the host cell by
the AAV
vector. Transduction is accomplished, following preparation of mature virions
containing the AAV vectors, by infection of HSC or progenitor cells therewith.
Transduced cells may be located in patients or transduced ex vivo and
introduced or
reintroduced into patients, e.g., by intravenous transfusion (Rosenberg,
1990).
In ex vivo embodiments, HSC or progenitor cells are provided by obtaining
bone marrow cells from patients and optionally enriching the bone marrow cell
population for HSC. HSC can be transduced by standard methods of transfection
or
infected with mature virions for about 1 to 2 hours at about 37°C.
Stable integration
of the viral genome is accomplished by incubation of HSC at about 37°C
for about
one week to about one month. The stable, site-specific integration and
erythroid cell-
specific expression is assessed as described above. After the transduced cells
have
been introduced into a patient, the presence of the heterologous gene product
can be
monitored or assessed by an appropriate assay for the gene product in the
patient, for
example in peripheral red blood cells or bone marrow of the patient when
expression
is erythroid cell-specific. As described above, the specific assay is
dependent upon
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
the nature of the heterologous gene product and can readily be determined by
one
skilled in the art.
a. Combined Therapy
5 The present invention has described methods of increasing the efficiency of
AAV-mediated gene transcription by providing expression constructs encoding
the
therapeutic gene of interest in combination with an FGFR and/or cell surface
HSPG.
Efficient gene transfer by AAV has been attributed the presence of an FGFR co-
receptor to mediate the uptake of the AAV once it has bound to the HSPG. Thus,
10 efficient transcription may be achieved by providing the expression
construct
containing the therapeutic transgene in combination with an expression
construct
containing an FGFR receptor or an HSPG receptor or both. Alternatively, the
host
receptors) may be stimulated, upregulated or otherwise encouraged to take-up
AAV
by provision of a stimulator of these receptors in combination with the
transgene
15 expression construct.
To stimulate the endogenous receptors or provide for the expression of such
receptors in the target cells, using the methods and compositions of the
present
invention, one would generally contact a "target" cell with the stimulator or
20 expression construct and contact the cell with the therapeutic transgenic
construct
(gene therapy). These compositions would be provided in a combined amount
effective to increase the AAV infection efficiency. This process may involve
contacting the cells with the gene therapy and the receptor encoding
constructs at the
same time. This may be achieved by contacting the cell with a single
composition that
25 includes both the therapeutic gene and the receptor gene(s), or by
contacting the cell
with two distinct compositions at the same time, wherein one composition
includes
the therapeutic expression construct and the other includes the receptor
expression
construct.
30 In addition to ensuring an efficient uptake of the AAV, it may be
beneficial to
provide agents or factors suitable for use in a combined therapy with the
therapeutic
expression construct.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
51
The inventors propose that the local or regional delivery of vector constructs
to
patients in need of gene therapy in combination with the receptor expression
will be a
very efficient method for delivering a therapeutically effective gene to
counteract the
clinical disease. Alternatively, systemic delivery of therapeutic expression
construct
may be appropriate in certain circumstances, for example, where extensive
metastasis
has occurred.
K. Formulations and Routes for Administration to Patients
Where clinical applications are contemplated, it will be necessary to prepare
pharmaceutical compositions - expression vectors, virus stocks, proteins,
antibodies
and drugs - in a form appropriate for the intended application. Generally,
this will
entail preparing compositions that are essentially free of pyrogens, as well
as other
impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render
delivery vectors stable and allow for uptake by target cells. Buffers also
will be
employed when recombinant cells are introduced into a patient. Aqueous
compositions of the present invention comprise an effective amount of the
vector to
cells, dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous
medium. Such compositions also are referred to as inocula. The phrase
"pharmaceutically or pharmacologically acceptable" refer to molecular entities
and
compositions that do not produce adverse, allergic, or other untoward
reactions when
administered to an animal or a human. As used herein, "pharmaceutically
acceptable
carrier" includes any and all solvents, dispersion media, coatings,
antibacterial and
antifungal agents, isotonic and absorption delaying agents and the like. The
use of
such media and agents for pharmaceutically active substances is well know in
the art.
Except insofar as any conventional media or agent is incompatible with the
vectors or
cells of the present invention, its use in therapeutic compositions is
contemplated.
Supplementary active ingredients also can be incorporated into the
compositions.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
52
The active compositions of the present invention may include classic
pharmaceutical preparations. Administration of these compositions according to
the
present invention will be via any common route so long as the target tissue is
available
via that route. This includes oral, nasal, buccal, rectal, vaginal or topical.
Alternatively, administration may be by orthotopic, intradermal, subcutaneous,
intramuscular, intraperitoneal or intravenous injection. Such compositions
would
normally be administered as pharmaceutically acceptable compositions,
described
supra.
The active compounds also may be administered parenterally or
intraperitoneally. Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water suitably mixed
with a
surfactant, such as hydroxypropylcellulose. Dispersions also can be prepared
in
glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under
ordinary
conditions of storage and use, these preparations contain a preservative to
prevent the
growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous
solutions or dispersions and sterile powders for the extemporaneous
preparation of
sterile injectable solutions or dispersions. In all cases the form must be
sterile and
must be fluid to the extent that easy syringability exists. It must be stable
under the
conditions of manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and fungi. The
carrier can
be a solvent or dispersion medium containing, for example, water, ethanol,
polyol (for
example, glycerol, propylene glycol, and liquid polyethylene glycol, and the
like),
suitable mixtures thereof, and vegetable oils. The proper fluidity can be
maintained,
for example, by the use of a coating, such as lecithin, by the maintenance of
the
required particle size in the case of dispersion and by the use of
surfactants. The
prevention of the action of microorganisms can be brought about by various
antibacterial an antifungal agents, for example, parabens, chlorobutanol,
phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be preferable to
include
isotonic agents, for example, sugars or sodium chloride. Prolonged absorption
of the
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
53
injectable compositions can be brought about by the use in the compositions of
agents ,
delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active
compounds
in the required amount in the appropriate solvent with various of the other
ingredients
enumerated above, as required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized active
ingredients into
a sterile vehicle which contains the basic dispersion medium and the required
other
ingredients from those enumerated above. In the case of sterile powders for
the
preparation of sterile injectable solutions, the preferred methods of
preparation are
vacuum-drying and freeze-drying techniques which yield a powder of the active
ingredient plus any additional desired ingredient from a previously sterile-
filtered
solution thereof.
As used herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal agents,
isotonic and
absorption delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except insofar as
any
conventional media or agent is incompatible with the active ingredient, its
use in the
therapeutic compositions is contemplated. Supplementary active ingredients
also can
be incorporated into the compositions.
For oral administration the polypeptides of the present invention may be
incorporated with excipients and used in the form of non-ingestible
mouthwashes and
dentifrices. A mouthwash may be prepared incorporating the active ingredient
in the
required amount in an appropriate solvent, such as a sodium borate solution
(Dobell's
Solution). Alternatively, the active ingredient may be incorporated into an
antiseptic
wash containing sodium borate, glycerin and potassium bicarbonate. The active
ingredient may also be dispersed in dentifrices, including: gels, pastes,
powders and
slurries. The active ingredient may be added in a therapeutically effective
amount to a
paste dentifrice that may include water, binders, abrasives, flavoring agents,
foaming
agents, and humectants.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
54
The compositions of the present invention may be formulated in a neutral or
salt form. Pharmaceutically-acceptable salts include the acid addition salts
(formed
with the free amino groups of the protein) and which are formed with inorganic
acids
such as, for example, hydrochloric or phosphoric acids, or such organic acids
as
acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free
carboxyl
groups also can be derived from inorganic bases such as, for example, sodium,
potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner compatible with
the dosage formulation and in such amount as is therapeutically effective. The
formulations are easily administered in a variety of dosage forms such as
injectable
solutions, drug release capsules and the like. For parenteral administration
in an
aqueous solution, for example, the solution should be suitably buffered if
necessary
and the liquid diluent first rendered isotonic with sufficient saline or
glucose. These
particular aqueous solutions are especially suitable for intravenous,
intramuscular,
subcutaneous and intraperitoneal administration. In this connection, sterile
aqueous
media which can be employed will be known to those of skill in the art in
light of the
present disclosure. For example, one dosage could be dissolved in 1 ml of
isotonic
NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected
at the
proposed site of infusion, (see for example, "Remington's Pharmaceutical
Sciences"
15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will
necessarily occur depending on the condition of the subject being treated. The
person
responsible for administration will, in any event, determine the appropriate
dose for
the individual subject. Moreover, for human administration, preparations
should meet
sterility, pyrogenicity, general safety and purity standards as required by
FDA Office
of Biologics standards.
L. Examples
The following examples are included to demonstrate preferred embodiments
of the invention. It should be appreciated by those of skill in the art that
the
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
techniques disclosed in the examples which follow represent techniques
discovered by
the inventor to function well in the practice of the invention, and thus can
be
considered to constitute preferred modes for its practice. However, those of
skill in
the art should , in light of the present disclosure, appreciate that many
changes can be
5 made in the specific embodiments which are disclosed and still obtain a like
or similar
result without departing from the concept, spirit and scope of the invention.
More
specifically, it will be apparent that certain agents which are both
chemically and
physiologically related may be substituted for the agents described herein
while the
same or similar results would be achieved. All such similar substitutes and
10 modifications apparent to those skilled in the art are deemed to be within
the spirit,
scope and concept of the invention as defined by the appended claims.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
56
EXAMPLE 1
Materials and Methods
Cells, plasmids, and viruses.
The human cervical carcinoma cell line HeLa, the human adenovirus-
transformed human embryonic kidney cell line 293, and murine fibroblast NIH3T3
cells were obtained from the American Type Culture Collection (Rockville, MD).
The human naso-pharyngeal carcinoma cell line KB, the human lymphoblastoid
cell
line Raji, and the human megakaryocytic leukemia cell line M07e, were obtained
respectively from Drs. Asok C. Antony, Zacharie A. Brahmi, and Hal E.
Broxmeyer
(Indiana University School of Medicine, Indianapolis, IN). Monolayer cultures
of
HeLa, KB, 293, and NIH3T3, and suspension cultures of M07e and Raji were
maintained in Iscove's-modified Dulbecco's medium (IMDM) supplemented with 10%
fetal bovine serum (FBS) and 1 % antibiotics. The recombinant murine HSPG
(Syndecan-1) (Saunders et al., 1989) was obtained from Dr. Bradley B. Olwin,
University of Colorado, Boulder, CO. The recombinant plasmid pSV7d/hFGFRI
containing the SV40 promoter-driven cDNA for human fibroblast growth factor
receptor 1 (FGFR1) has been described previously (Johnson et al., 1990), and
was
obtained from Dr. Lewis T. Williams (University of California, San Francisco,
CA).
Recombinant AAV plasmid pCMVp-lacZ containing the CMVp-driven b-
galactosidase (lacZ) gene has been described elsewhere (Ponnazhagan et al.,
1997,
Ponnazhagan et al., 1997). Wild-type (wt) and recombinant AAV vector (vCMVp-
lacZ) stocks were generated and purified by CsCI equilibrium density gradient
centrifugation as previously described (Ponnazhagan et al., 1997, Qing et al.,
1997,
Qing et al., 1998, Ponnazhagan et al., 1997, Ponnazhagan et al., 1997).
Physical
particle titers of wt and recombinant vector stocks were determined by
quantitative
DNA slot blot analysis (Kube and Srivastava, 1997). Physical
particle:infectious
particle ratio (approximately 1000:1), and the contaminating wild-type AAV-
like
particle titer (approximately 0.01 %) in the recombinant vector stocks were
determined
as previously described (Wang et al., 1998).
CA 02358094 2001-06-28
WO 00/39311 PCTNS99/31220
57
AAV-binding assay.
AAV-binding studies were carried out as previously described (Qing et al.,
1998, Ponnazhagan et al., 1997). Briefly, 5 x 104 cells were washed twice with
IMDM containing 1 % BSA. One ml of IMDM containing 1 % BSA was added to the
cells with either 1.5 x108 particles of [35S] methionine-labeled wt AAV alone
or with
100-fold excess of unlabeled wt AAV particles for 90 min. at 4~C. Following
incubation, cells were washed four times with IMDM containing 1 % BSA and
solubilized with 1 ml 0.5 N NaOH for 30 min at room temperature. Radioactivity
of
lysates was determined and specific binding was calculated as the total
radioactivity
minus the non-specific radioactivity as previously described (Qing et al.,
1998).
FGF-binding assay.
FGF-binding experiments were carried out as previously described by Kan et
al. (1993) with the following modifications. Briefly, 5 x 104 cells were
washed twice
with IMDM containing 0.1 % BSA. One ml of IMDM containing 0.1 % BSA was
added to all cells either with 0.5 ng/ml l2sl_bFGF obtained from Amersham
(Arlington Heights, IL) alone or with large excess unlabeled bFGF (Sigma
Chemical
Co., St. Louis, MO). Cells were incubated for 90 min. at room temp. Following
incubation, cells were loaded on Whatman GF/C glass fiber filters and washed
four
times with IMDM containing 0.1 % BSA to remove free lzsl-bFGF from cell-
associated bFGF. Radioactivity of lysates was determined in a Beckman Gamma
counter. Specific binding was calculated as the total radioactivity minus the
non-
specific radioactivity.
Stable transfection with HSPG and/or FGFRl expression plasmids.
Transfection of M07e and Raji cells with different expression plasmid DNAs
was carried out using the DMRIE-C reagent according to the protocol provided
by the
vendor (Gibco-BRL, Grand Island, NY). Selectable marker genes (HygR and Neon)
were inserted into HSPG and FGFR1 cDNA expression plasmids, respectively, by
standard cloning methods. Either 6418, hygromycin, or both, were added at a
final
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
58
conc. of 400 mg/ml and 300 mg/ml, respectively, 48 hrs post-transfection, and
individual drug-resistant cell clones were isolated after 28 days of
selection.
Recombinant AAV transduction assays.
Approximately equivalent numbers of cells were washed once with IMDM
and then infected with the recombinant vCMVp-lacZ vector at various indicated
particles/cell. Forty-eight h post-infection (p.i.), cells were either fixed
and stained
with X-gal (5-bromo-4-chloro-3-indolyl b-D-galactopyranoside) and the numbers
of
blue cells were enumerated, or analyzed by FACS as previously described
(Ponnazhagan et al., 1997, Qing et al., 1997, Qing et al., 1998, Ponnazhagan
et al.,
1997, Ponnazhagan et al., 1997). In some studies, following infection with the
recombinant vCMVp-lacZ vector, cells were washed extensively with PBS, and low
Mr DNA samples isolated from equivalent numbers of cells were analyzed on
Southern blots using the lacZ DNA probe as described previously (Ponnazhagan
et
al., 1997).
Cellular tyrosine kinase inhibitors and treatment conditions.
Specific inhibitors of cellular protein tyrosine kinases (genistein; Sigma
Chemical Co., St. Louis, MO), of EGFR PTK (tyrphostin l; Sigma), and FGFR PTK
(SU4989 and SU5402; Calbiochem, La Jolla, CA), were dissolved in
dimethylsulphoxide (DMSO), and stock solutions were stored at 4~C and diluted
in
1MDM prior to use in experiments. Cells were either mock-treated or treated
with
various concentrations of these compounds separately for 2 hrs at 37°C.
Following
treatments, cells were washed twice with PBS and were either mock-infected or
infected with the recombinant AAV vector as described above.
EXAMPLE 2
Successful Infection of Cells by AAV Requires a Cell Surface Co-
Receptor
All previously published studies have established that cell types that can
bind
AAV can be infected by AAV (Qing et al., 1998, Summerford and Samulski, 1998,
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
59
Ponnazhagan et al., 1996). For example, although the transduction efficiency
varies
greatly, permissive human cells, such as HeLa, KB, and 293, can bind AAV, but
non-
permissive cells, such as M07e, can not (FIG. lA). Interestingly, however, the
inventors noted that murine NIH3T3 cells could also bind AAV efficiently, but
could
not be transduced by a recombinant AAV vector containing the cytomegalovirus
immediate-early promoter-driven b-galactosidase gene (vCMVp-lacZ) (FIG. 1B-
FIG.
lE). Since the efficiency of AAV-mediated transgene expression in various cell
types
is dependent upon the phosphorylation status of the cellular ssD-BP (Qing et
al.,
1998), the inventors performed transduction studies, under identical
conditions, with
HeLa and NIH3T3 cells, with or without prior treatment with tyrphostin 1,
previously
shown to augment AAV-mediated transgene expression (Mah et al., 1998). These
results are depicted in FIG. 1B-FIG. lE. As can be seen, whereas the low-level
of
transgene expression in untreated HeLa cells (FIG. 1B) could be significantly
increased following treatment with tyrphostin 1 (FIG. 1C), as observed
previously
(Mah et al., 1998), transgene expression could not be detected in NIH3T3 cells
(FIG.
1D), and tyrphostin 1 treatment failed to elicit a significant response in
these cells
(FIG. lE). The lack of this response in NIH3T3 cells was not due the failure
of
tyrphostin 1 treatment to catalyze dephosphorylation of the ssD-BP since in
both cell
types, predominantly the dephosphorylated form of the ssD-BP was present
following
treatment with tyrphostin l, as detected by electrophoretic mobility-shift
assays
(EMSA) (Qing et al., 1997, Qing et al., 1998). These results led the inventors
to
hypothesize that following initial attachment of AAV to the cell surface via
the HSPG
receptor, efficient entry of the virus requires the presence of a putative
cellular co-
receptor.
EXAMPLE 3
Fibroblast Growth Factor Receptor 1 (FGFRl) is Required for AAV
Binding
Given the fact that HSPG is not sufficient for AAV entry into a cell, the
inventors began the search for the co-receptor required for AAV infection. The
inventors reasoned that AAV might utilize FGFR as a co-receptor for entry into
the
host cell. To this end, M07e cells, known to be non-permissive for AAV
infection
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
(Ponnazhagan et al., 1996), were stably transfected with the following
expression
plasmids, either alone, or in combination: murine (mu) HSPG; and human (hu)
FGFR1. An additional human lymphoblastoid cell line, Raji, known to be
negative
for cell surface expression both of HSPG and FGFR (Ixbakken and Rapraeger,
1996,
5 Kiefer et al., 1990), was also stably transfected with muHSPG, huFGFRI, or
both.
Mock-transfected and three individual clones each from M07e and Raji cells
were
analyzed separately for binding of l2sl-bFGF and 35S-AAV as previously
described
(Qing et al., 1998, Mah et al., 1998, Kan et al., 1993). These results are
shown in
FIG. 2A and FIG. 2B. It is interesting to note that M07e cells, known to lack
HSPG
10 expression (Barlett and Samulski, 1998), fail to bind bFGF, but following
stable
transfection with muHSPG cDNA, allow significant binding of bFGF. A low-level
of
bFGF binding also occurs in M07e cells stably transfected with huFGFRl cDNA
alone, the extent of which is significantly higher when M07e cells co-express
HSPG
and FGFR1 (FIG. 2A). These results suggest that M07e cells do indeed express
the
15 endogenous FGFR gene. Mock-tranfected Raji cells also fail to bind bFGF, as
expected, and only low-levels of bFGF binding are detected in Raji cells
stably
transfected with either the HSPG or the FGFR1 expression plasmid alone. In
Raji
cells that co-express HSPG+FGFR1, significant binding of bFGF occurs further
corroborating the requirement of both HSPG and FGFR1 for the ligand binding.
It
20 also is interesting that the binding patterns of radiolabled AAV to these
two cell types
closely resemble that of bFGF binding (FIG. 2B). Taken together, these data
strongly
suggest that cell surface expression both of HSPG and FGFR1 is required for
successful binding of AAV to the host cell.
25 EXAMPLE 4
Cell Surface Co-expression of HSPG and FGFRl Confers AAV Infectivity
to Non-permissive Cells.
It was next of interest to examine whether M07e and Raji cells stably
transfected with HSPG, or FGFR1, or both, could be successfully transduced
with the
30 recombinant vCMVp-lacZ vector. Individual clonal isolates from both cell
types were
either mock-infected or infected with vCMVp-lacZ under identical conditions
and
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
61
analyzed for transgene expression by fluorescence-activated cell sorting
(FACS).
Representative histograms of each of these clones are shown in FIG. 3A-FIG.
3H.
It is evident that, whereas little transgene expression occurred in mock-
s infected M07e cells as expected, M07e cells expressing either HSPG alone, or
co-
expressing HSPG+FGFRI, but not FGFR1 alone, could be readily transduced by the
recombinant AAV vector (FIG. 3A-FIG. 3D). Because M07e cells express the
endogenous FGFR gene, expression of the exogenous HSPG is sufficient to render
these cells permissive to AAV infection. Raji cells, on the other hand, fail
to allow
AAV-mediated transduction if only the exogenous HSPG or FGFR1 genes are
expressed, but co-expression of HSPG+FGFR1 genes confers, albeit at a
relatively
low-efficiency, AAV infectivity to these cells (FIG. 3E-FIG. 3H). The
underlying
mechanism of low-efficiency of transduction in Raji cells was investigated
further by
examining the extent of the viral DNA entry as described in Methods. It is
evident
that despite similar levels of viral binding (FIG. 2B), the extent of the
viral DNA entry
into Raji cells co-expressing HSPG+FGFR1 was significantly lower than that in
M07e
cells which correlated well with AAV transduction efficiencies in the two cell
types.
Additional individual clonal isolates from both cell types were also evaluated
for
AAV transduction efficiency. The cumulative data are presented in Table 3. It
is
evident that M07e and Raji cell clones co-expressing FGFR1+HSPG are transduced
at
an average transduction efficiency of approximately 70% and 10%, respectively.
Taken together, these studies establish that co-expression of HSPG+FGFR1 is
required not only for binding to, but also for entry of, AAV into the host
cell.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
62
Table 3. Effect of stable transfection of M07e and Raji cells with the various
indicated expression plasmid DNAs on AAV-mediated transgene
expression.
CELL CLONES Percent cells
expressing
the lacZ
gene
+muHSPG +huFGFRl +muHSPG+huFGFRl
M07e Clone 1 67.1 2.7 74.5
M07e Clone 2 65.9 2.7 75.6
M07e Clone 3 69.5 2.0 74.9
Raji Clone 1 2.9 0.1 9.5
Raji Clone 2 3.5 0.1 9.8
Raji Clone 3 2.5 0.2 10.4
EXAMPLE 5
AAV-mediated Transgene Expression Requires
Dephosphorylation of the Cellular ssD-BP.
The inventors have recently provided evidence that a host cell protein,
designated the single-stranded D-sequence-binding protein (ssD-BP), is a
crucial
determinant of AAV transduction efficiency (Qing et al., 1997; Qing et al.,
1998).
The inventors have also determined that the ssD-BP is phosphorylated at
tyrosine
residues by the protein tyrosine kinase (PTK) activity of the cellular
epidermal growth
factor receptor (EGFR) since treatment of cells with tyrphostins, specific
inhibitors of
the EGFR PTK, causes dephosphorylation of the ssD-BP and leads to significant
augmentation in AAV-mediated, post-receptor transgene expression (Mah et al.,
1998). In order to evaluate whether the same mechanism was operational in M07e
and Raji cells, these cell types stably co-transfected with HSPG and FGFRl
expression plasmids were either mock-infected, or infected with the
recombinant
vCMVp-lacZ vector, either in the presence of co-infection with human
adenovirus 2
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
63
(Ad2), or with prior treatment with tyrphostin 1, as previously described (Mah
et al.,
1998) since the ssD-BP in both cell types is present predominantly in the
phosphorylated state and AAV-mediated transduction efficiency is approximately
2-
3%. Transgene expression was analyzed by FACS as described above. These
results
are shown in FIG. 4A-FIG. 4F. It is interesting to note that whereas little
transgene
expression occurred in mock-infected M07e (FIG. 4A) and Raji (FIG. 4D) cells,
as
expected, each of the cell types co-expressing HSPG+FGFR1 exhibited roughly
the
same efficiency of transgene expression either in the presence of Ad2 or with
prior
treatment with tyrphostin 1. These studies were extended to include additional
individual clonal isolates from both cell types. The cumulative data,
presented in
Table 4, further corroborate that post-receptor entry, dephosphorylation of
the ssD-BP
is necessary to allow AAV-mediated transgene expression.
Table 4. Relative effects of co-infection with human adenovirus 2 or treatment
with Tyrphostin 1 on AAV-mediated transgene expression in M07e
and Raji cell clones stably co-transfected with muHSPG+huFGFRl
expression plasmid DNAs.
Cell clones Percent cells expressing
the lacZ gene
(+muHSPG+huFGFRl)
+Adenovirus 2 +Tyrphostin 1
M07e Clone 1 81.7 82.3
M07e Clone 2 81.8 81.6
M07e Clone 3 81
7
. 83.8
Raji Clone 1 20.6 23.0
Raji Clone 2 25.7 22.9
Raji Clone 3 25.5 26.8
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
64
EXAMPLE 6
FGFR Autophosphorylation is not
Required for AAV-mediated Transduction
Since ligand binding to FGFR leads to receptor dimerization followed by
receptor autophosphorylation leading to recruitment of intracellular signaling
molecules (Rapraeger et al., 1991, Ledoux et al., 1992, Roghani and
Moscatelli, 1992,
Givol and Yayon, 1992, Kan et al., 1993), it was next of interest to determine
whether
the FGFR-associated protein tyrosine kinase (PTK) activity affected AAV-
mediated
transgene expression. Human 293 and HeLa cells, known to be permissive for AAV
infection, were either mock-treated, or first treated with specific inhibitors
of FGFR
PTK activity (Strawn et al., 1996, Mohammadi et al., 1997) followed by
infection
with the recombinant vCMVp-lacZ vector under identical conditions and the
extent of
transgene expression was determined 48 hours post infection as described
above. The
results of these studies indicated that none of the FGFR PTK inhibitors had
any
significant effect on AAV-mediated transgene expression. The inventors
conclude
from these studies that FGFR PTK activity is not required for AAV-mediated
transgene expression.
EXAMPLE 7
FGF Treatment Perturbs AAV Binding to Non-permissive and Permissive
Cells and Abrogates Viral entry into Permissive Cells.
In order to further corroborate the involvement of FGFR1 as a co-receptor for
AAV binding and/or entry, the inventors reasoned that treatment of non-
permissive
cells, such as NIH3T3 cells, and permissive cells, such as 293 cells, with
large excess
of bFGF during AAV infection would perturb AAV binding and infection,
respectively. To this end, the following two sets of studies were carried out.
In the
first set, binding studies were carried out with radiolabeled AAV using NIH3T3
and
293 cells in the absence or presence of large excess of bFGF. Several
additional
controls that included unlabeled wt AAV, or heparin (as positive controls),
and EGF
(as a negative control), were also included. The results of such a study are
shown in
FIGS. 6-A and 6-B. It is evident that AAV binding to NIH3T3 cells was
inhibited by
heparin, as expected (Summerford and Samulski, 1998), bFGF also inhibited AAV
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
binding to a significant extent, whereas EGF had no effect under identical
conditions.
Similarly, unlabeled wt AAV significantly inhibited binding of radiolabeled
AAV to
293 cells, as expected. AAV binding to 293 cells was also reduced in the
presence of
bFGF. Similar concentrations of EGF, on the other hand, had no significant
effect on
5 AAV binding to 293 cells. In the second set of studies, equivalent numbers
of 293
cells were transduced with the recombinant vCMVp-lacZ vector either in the
absence
or presence of large excess of bFGF or EGF under identical conditions.
Transgene
expression was evaluated 48 h post-transduction by X-gal staining as
previously
described (Ponnazhagan ~et al., 1997). The results of these studies are shown
in
10 FIG. SC-FIG. SH. It was determined that AAV-mediated transduction of 293
cells
(FIG. SC) was inhibited by approximately 89% in the presence of bFGF (FIG.
SE), but
only by approximately 2% in the presence of EGF (FIG. SG). These assays,
carried
out with 293 cells with prior treatment with genistein (FIG. SD, FIG. SF and
FIG. SH)
a specific inhibitor of cellular protein tyrosine kinases, known to augment
AAV
15 transduction efficiency (Qing et al., 1997), yielded similar results (~89%
inhibition
with bFGF; 0% inhibition with EGF) indicating that lack of transgene
expression in
the presence of bFGF (FIG. SF) was not due to phosphorylation of the ssD-BP in
293
cells. Taken together, these results firmly establish that the HSPG-FGFRI
interaction
is crucial not only for successful binding, but also for entry of AAV into
cells.
EXAMPLE 8
AAV Co-receptor Activity of FGFRl, FGFR2, FGFR3, and FGFR4.
Experiments in previous examples were carried out with the FGFR 1 cDNA
under the control of the SV40 promoter. There are at least three additional
distinct but
related members in the FGFR family, viz. FGFR2, FGFR3, and FGFR4 (Hughes,
1997), which are differentially expressed in primary human tissues. FGFR1 is
expressed abundantly in brain neurons and cardiac myocytes, whereas FGFR2
expression predominates in the choroid plexus and glial cells. FGFR3
expression
abounds in the intestine and growth plates, and FGFR4 is expressed in
hepatocytes
and the adrenal glands (Gonzalez et al., 1996; Ozawa et al., 1996; Olwin et
al., 1989).
The amino acid sequence within the intracellular kinase domain is highly
variable
among the different FGFRs (14-79% homology), and perhaps accounts for their
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
66
different signaling and mitogenic potentials (Wang et al., 1994). The
inventors
obtained FGFR1, FGFR2, FGFR3, and FGFR4 cDNA expression plasmids, each
under the control of the CMV promoter, from Dr. Dan Donoghue, University of
California, San Diego. Each of the plasmids was transfected into control and
HSPG-
expressing Raji cells, and clonal populations were analyzed for the various
types of
FGFRs for their potential co-receptor activity for AAV-mediated transduction.
These
results are shown in Figure 7. FGFRI possesses the highest activity, followed
by
FGFR2 and FGFR3. In these experiments, the inventors were not able to detect
any
co-receptor activity with FGFR4.
All of the compositions and/or methods disclosed and claimed herein can be
made and executed without undue experimentation in light of the present
disclosure.
While the compositions and methods of this invention have been described in
terms of
preferred embodiments, it will be apparent to those of skill in the art that
variations
may be applied to the compositions andlor methods and in the steps or in the
sequence
of steps of the method described herein without departing from the concept,
spirit and
scope of the invention. More specifically, it will be apparent that certain
agents which
are both chemically and physiologically related may be substituted for the
agents
described herein while the same or similar results would be achieved. All such
similar substitutes and modifications apparent to those skilled in the art are
deemed to
be within the spirit, scope and concept of the invention as defined by the
appended
claims.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
67
REFERENCES
The following references, to the extent that they provide exemplary procedural
or other details supplementary to those set forth herein, are specifically
incorporated
herein by reference.
Alkhatib et al. Science 272, 1955-1958 (1996).
Anderson et al. Nature, 332:360, 1988
Arap et al., Cancer Res., 55:1351-1354, 1995.
Baichwal and Sugden, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum
Press, pp. 117-148, 1986.
Bartlett, and Samulski, Nature Med. 4, 635-637 (1998).
Basilico and Moscatelli, Adv. Cancer Res. 59, 115, 1992
Benvenisty and Neshif, Proc. nat'l. acad. sci. USA, 83:9551-9555, 1986.
Bergelson et al. Science 275, 1320-1323 (1997).
Berns and Bohenzky, Adv. Virus Res., 32:243-307, 1987.
Berns and Giraud, Curr. Top. Microbiol. Immunol., 218:1-23, 1996.
Berns, Parvoviridae: the viruses and their replication, p. 2173-2197. In B. N.
Fields,
D. M. Knipe, and P. M. Howley (ed.), Fields virology, vol. 3. Lippincott-
Raven, Philadelphia, Pa., 1996
Bertran et al., J. Virol., 70 (10) 6759-6766, 1996.
Burgess and Kelly, Annu. Rev. Cell. Biol. 3:243-293, 1987.
Bussemakers et al., Cancer Res., 52:2916-2922, 1992.
Caldas et al., Nat. Genet., 8:27-32, 1994.
Carter and Flotte, Curr. Top. Microbiol. Immunol., 218:119-144, 1996.
Carter, Curr. Opin. Biotechnol., 3:533-539, 1992.
Casey et al, Oncogene, 6:1791-1797, 1991.
Chatterjee and Wong Jr., Curr. Top. Microbiol. Immunol., 218:61-73, 1996.
Chatterjee, et al., Ann. N. Y. Acad. Sci., 770:79-90, 1995.
Chavez et al., In: Methods in Cell Biology, Roth, M., Ed. New York, Academic
Press,
43:263-288, 1994.
Chen and Okayama, Mol. Cell Biol., 7:2745-2752, 1987.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
68
Chen et al., Nat. Med. 3:866-871, 1997
Cheng et al., Cancer Res., 54:5547-5551, 1994.
Cheung et al., Biochem. J., 295:427-435, 1993c.
Cheung et al., J. Biol. Chem., 268:24303-24310, 1993a.
Cheung et al., J. Biol. Chem., 268:6139-6146, 1993b.
Coffin, In: Virology, Fields BN, Knipe DM, ed., New York: Raven Press, pp.
1437-
1500, 1990.
Compton et al., Virology 193:834-841, 1993.
Diertrich and Cassaro J. Biol. Chem. 252:2254-2261 1977
Dubensky et al., Proc. nat'l. acad. sci. USA, 81:7529-7533, 1984.
Edelman and Crossin, Annu. Rev. Biochem., 60:155-190, 1991
Edelman, Annu. Rev. Biochem., 54:135-169, 1985.
Fechheimer et al., Proc. Nat'l. Acad. Sci. USA, 84:8463-8467, 1987.
Ferber et al., Mol. Endocrinol. 5(3):319-26, 1991.
Ferkol et al., FASEB J., 7:1081-1091, 1993.
Ferrari et al., J. Virol., 70:3227-3234, 1996.
Fisher et al. J. Virol. 70, 520-532 (1996).
Fisher et al., Nat. Med., 3:306-312, 1997
Flotte and Carter, Gene Ther., 2:357-362, 1995.
Flotte et al., Proc. Nat'l. Acad. Sci. USA, 90:10613-10617, 1993.
Fraley et al., Proc. Nat'l. Acad. Sci. USA, 76:3348-3352, 1979.
Freshner, In: Animal Cell Culture: a Practical Approach, Second Edition,
Oxford/New York, IRL Press, Oxford University Press, 1992.
Frixen et al., J. Cell Biol., 113:173-185, 1991.
Geraghty et al., Science 280, 1618-1620 (1998).
Gosh and Bachhawat, In: Liver diseases, targeted diagnosis and therapy using
specific receptors and ligands, Wu G, Wu C ed., New York: Marcel Dekker,
pp. 87-104, 1991.
Giancotti and Ruoslahti, Cell, 60:849-859, 1990.
Givol and Yayon, FASEB J. 6, 3362-3369 ( 1992).
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
69
Gonzalez, Hill, Logan, Maher and Baird, "Distribution of fibroblast growth
factor
(FGF)-2 and FGF receptor-1 messenger RNA expression and protein presence
in the mid-trimester human fetus," Pediatr. Res., 39: 375, 1996.
Goodman et al., Blood, 84:1492-1500, 1994.
Gopal, Mol. Cell Biol., 5:1188-1190, 1985.
Gospodarowicz et al., Mol. Cell. Endocrinol., 46:107, 1986
Graham and Van Der Eb, Virology, 52:456-467, 1973.
Guillonneau et al., Mol Biol Cell. 9( 10):2785-802, 1998.
Harland and Weintraub, J. Cell Biol., 101:1094-1099, 1985.
He, et al. Nature 385, 645-649 (1997).
Hollestein et al., Science, 253:49-53 1991.
Hughes, "Differential expression of the fibroblast growth factor receptor
(FGFR)
multigene family in normal human adult tissues," J. Histochem. Cytochem.,
45: 1005, 1997.
Hussussian et al., Nature Genetics, 15-21, 1994.
Ishihara et al., J. Biol. Chem. 268, 4675; 1993
Jackson et al., J. Virol. 70:5282-5287, 1996
Jackson et al., Physiol. Rev. 71:481-539, 1991
Johnson et al., In: Biotechnology And Pharmacy, Pezzuto et al., eds., Chapman
and
Hall, New York, 1993.
Johnson et al., Mol. Cell. Biol. 10, 4728-4736 (1990).
Kamb et al., Nature Genetics, 8:22-26, 1994.
Kamb et al., Science, 2674:436-440, 1994.
Kan et al., Science 259, 1918-1921 (1993).
Kan et al., Science 259, 1918, 1993
Kaneda et al., Science, 243:375-378, 1989.
Kaplitt et al., Arm. Thor. Surg., 62:1669-1676, 1996.
Kaplitt et al., Nat. Genet., 8:148-153, 1994.
Kato et al., J. Biol. Chem., 266:3361-3364, 1991.
Kearns et al., Gene Ther., 3:748-755, 1996.
Kessler et al., Proc. Nat'l. Acad. Sci. USA, 93:14082-14087, 1996.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
Kiefer, et al. Proc. Nat'l. Acad. Sci., USA 87, 6985-6989 ( 1990).
Kjellen and Lindahl, Annu. Rev. Biochem. 60:443-475, 1991
Klagsbrun and Baird, Cell 67, 229, 1991
Klein et al., Nature, 327:70-73, 1987.
5 Koeberl et al., Proc. Nat'l. Acad. Sci. USA, 94:1426-1431, 1997.
Kotin et al., Genomics, 10:831-834, 1991.
Kotin et al., Proc. Nat'l. Acad. Sci. USA, 87:2211-2215, 1990.
Kube and Srivastava, Nucl. Acids Res., 25:3375-3376, 1997.
Kube et al., J. Virol., 71:7361-7371, 1997.
10 Laquerre, S. et al. J. Virol. 72, 6119-6130 (1998).
Lebakken, and Rapraeger, J. Cell Biol. 132, 1209-1221 (1996).
Ledoux et al., Prog. Growth Factor Res. 4, 107-120 (1992).
Lee et al., Science 245:57-60, 1989
Lin and Guidotti, J. Biol. Chem., 264:14408-14414, 1989.
15 Linhardt et al., Appl. Biochem. Biotechnol. 12:135-177, 1986
Macejak and Sarnow, Nature, 353:90-94, 1991.
Mah et al. J. Virol. 72, 9835-9843 (1998).
Mains et al., Mol. Endocrinol., 9:(1)3-13, 1995.
Mann et al., Cell, 33:153-159, 1983.
20 Marics et al., Oncogene 3:335, 1989.
Matsura et al., Brit. J. Cancer, 66:1122-1130, 1992.
McCown et al., Brain Res., 713:99-107, 1996.
Mercer, Critic. Rev. Eukar. Gene Express., 2:251-263, 1992.
Mettenleiter et al., J. Virol. 64:278-286, 1990
25 Mizukami et al., Virology, 217:124-130, 1996.
Mohammadi, et al. Science 276, 955-960 ( 1997).
Montenarh, Crit. Rev. Oncogen, 3:233-256, 1992.
Montgomery et al., Cell 87, 427-436 (1996).
Moore et al., EMBO. J., 5:919, 1986
30 Mori et al., Cancer Res., 54:3396-3397, 1994.
Muzyczka, Curr. Top. Microbiol. Immunol., 158:97-129, 1992.
Myers, EPO 0273085.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
71
Neufeld and D. Gospodarowicz, J. Biol. Chem., 261:5631, 1986.
Nicolas and Rubenstein, In: Vectors: A survey of molecular cloning vectors and
their
uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, pp. 493-513,
1988.
Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982.
Nicolau et al., Methods Enzymol., 149:157-176, 1987.
Nobri et al., Nature (London), 368:753-756, 1995.
Obrink, BioEssays., 13:227-233, 1991.
Odin and Obrink, Exp. Cell Res., 171:1-15, 1987.
Okamoto et al., Proc. Nat'l. Acad. Sci. USA, 91:11045-11049, 1994.
Olwin and Hauschka, "Cell type and tissue distribution of the fibroblast
growth factor
receptor," J. Cell. Biochem., 39: 443, 1989.
Orlow et al., Cancer Res., 54:2848-2851, 1994.
Ornitz et al., Mol. Cell. Biol. 12, 240, 1992
Ozawa, Uruno, Miyakawa, Seo, and Imamura, "Expression of the fibroblast growth
factor family and their receptor family genes during mouse brain
development," Brain Res. Mol. Brain Res., 41: 279, 1996.
Pelletier and Sonenberg, Nature, 334:320-325, 1988.
Perales et al., Proc. Nat'l. Acad. Sci. 91:4086-4090, 1994.
Ping et al., Microcirculation, 3:225-228, 1996.
Ponnazhagan et al., Gene, 190:203-210, 1997c.
Ponnazhagan et al., Hum. Gene Ther., 8:275-284. 1997a.
Ponnazhagan et al., J. Gen. Virol., 77:1111-1122, 1996.
Ponnazhagan et al., J. Virol., 71:3098-3104, 1997b.
Ponnazhagan et al., J. Virol., 71:8262-8267, 1997d.
Potter et al., Proc. Nat'l. Acad. Sci. USA, 81:7161-7165, 1984.
Qing et al., J. Virol., 71:5663-5667, 1997a.
Qing et al., J. Virol., 72:1593-1599, 1998.
Qing et al., Proc. Nat'l. Acad. Sci. USA, 94:10879-10884, 1997b.
Rapraeger et al., Science 252, 1705 1991
Ridgeway, In: Vectors: A survey of molecular cloning vectors and their uses,
Rodriguez
RL, Denhardt DT, ed., Stoneham: Butterworth, pp. 467-492, 1988.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
72
Rippe et al., Mol. Cell Biol., 10:689-695, 1990.
Roghani and Moscatelli, J. Biol. Chem. 267, 22156-22162, 1992.
Rosenberg, Duodecim., 106: ( 14) 1027-1029, 1990.
Rostand and Esko, Infect. Immun. 65:1-8, 1997.
Rubin et al., Proc. Nat'l. Acad. Sci. USA, 86:802 1989
Samulski et al., EMBO J., 10:3941-3950, 1991.
Samulski et al., J. Virol. 61(10):3096-3101, 1987.
Samulski et al., J. Virol., 36:3822-3828, 1989.
Saunders, et al., J. Cell Biol. 108, 1547-1556 (1989).
Serrano et al., Nature, 366:704-707, 1993.
Serrano et al., Science, 267:249-252, 1995.
Sheih et al., J. Cell Biol. 116:1273-1281, 1992
Srivastava et al., Cancer Genet Cytogenet., 35:(1)61-71, 1988.
Srivastava et al., Curr. Top. Microbiol. Immunol., 218:93-117, 1996.
Srivastava et al., J. Virol., 45:555-564, 1983.
Strawn, et al. Cancer Res. 56, 3540-3545 ( 1996).
Summerford and Samulski, J. Virol. 72, 1438-1445 (1998).
Taira et al., Proc. Nat'l. Acad. Sci. USA, 84:2980, 1987
Takahashi et al., Cancer Res., 52:2340-2342, 1992.
Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-
188,
1986.
Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986.
U.S. Patent 4,554,101.
U.S. Patent 4,933,294.
U.S. Patent 4,943,533.
U.S. Patent 5,183,884.
U.S. Patent 5,229,501
U.S. Patent 5,359,046.
U.S. Patent 5,378,809.
U.S. Patent 5,480,968.
U.S. Patent 5,487,979.
U.S. Patent 5,554,519.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
73
U.S. Patent 5,558,864.
U.S. Patent 5,587,459.
U.S. Patent 5,610,018.
U.S. Patent 5,610,288.
U.S. Patent 5,614,488.
U.S. Patent 5,654,307.
U.S. Patent 5,674,753.
U.S. Patent 5,679,683.
U.S. Patent 5,707,632
U.S. Patent 5,708,156.
U.S. Patent 5,717,067.
U.S. Patent 5,763,198.
U.S. Patent 5,773,476.
U.S. Patent 5,789,427.
Umbas et al., Cancer Res., 52:5104-5109, 1992.
Wagner et al., Proc. Nat'l. Acad. Sci. 87(9):3410-3414, 1990.
Walsh et al., J. Clin. Invest., 94:1440-1448, 1994.
Wang et al., J. Mol. Biol., 250:573-580, 1995.
Wang et al., J. Virol., 70:1668-1677, 1996.
Wang et al., J. Virol., 71:3077-3082, 1997.
Wang et al., J. Virol., 72:5472-5480, 1998.
Wang, Gao, and Goldfarb, "Fibroblast growth factor receptors have different
signaling
and mitogenic potentials," Mol. Cell. Biol., 14: 181, 1994.
Weinberg, Science, 254:1138-1146, 1991.
Werner et al., Mol Cell Biol. 12( 1 ):82-8, 1992.
Wong et al., Gene, 10:87-94, 1980.
Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993.
Wu and Wu, Biochemistry, 27:887-892, 1988.
Wu and Wu, J. Biol. Chem., 262:4429-4432, 1987.
WuDunn and Spear, J. Virol. 63:52-58, 1989
Xiao et al., J. Virol., 70:8098-8108, 1996.
CA 02358094 2001-06-28
WO 00/39311 PCT/US99/31220
74
Yang et al., Proc. Nat'l. Acad. Sci USA, 87:9568-9572, 1990.
Yayon et al., Cell 64, 841, 1991
Zelenin et al., FEBS Lett., 280:94-96, 1991.
Zhan et al., Mol. Cell. Biol., 8:3487 1988;
Zhou et al., Gene Ther., 3:223-229, 1996.
