U.S. patent application number 11/829313 was filed with the patent office on 2008-02-21 for gene transfer methods.
This patent application is currently assigned to Takara Shuzo Co., Ltd.. Invention is credited to Kiyozo Asada, Hideto Chono, Kei Fujinaga, Kimikazu Hashino, Ikunoshin Kato, Haruko Konishi, Tsuyoshi Miyamura, Mio Morishita, Mutsumi Sano, Mitsuhiro UENO, Hirofumi Yoshioka.
Application Number | 20080044903 11/829313 |
Document ID | / |
Family ID | 26397921 |
Filed Date | 2008-02-21 |
United States Patent
Application |
20080044903 |
Kind Code |
A1 |
UENO; Mitsuhiro ; et
al. |
February 21, 2008 |
GENE TRANSFER METHODS
Abstract
Improved methods for transferring a gene into target cells by
using a retrovirus, wherein the gene transfer efficiency is
improved and the target cells are efficiently transformed by
binding the retrovirus to a functional substance which is
immobilized on as carrier and having an activity of binding to
retroviruses followed by washing; using an antibody capable of
specifically recognizing cells, laminin or mannose-rich type sugar
chain as a substance having an activity of binding to the target
cells; pre-treating the target cells so as to inactivate
transferring receptor, or introducing a new functional group into
the functional substance.
Inventors: |
UENO; Mitsuhiro;
(Kasatsu-shi, JP) ; Yoshioka; Hirofumi;
(Kasatsu-shi, JP) ; Konishi; Haruko; (Kyoto-shi,
JP) ; Hashino; Kimikazu; (Osaka, JP) ;
Morishita; Mio; (Otsu-shi, JP) ; Chono; Hideto;
(Moriyama-shi, JP) ; Miyamura; Tsuyoshi;
(Kusatsu-shi, JP) ; Sano; Mutsumi; (Otsu-shi,
JP) ; Asada; Kiyozo; (Koka-gun, JP) ;
Fujinaga; Kei; (Otsu-shi, JP) ; Kato; Ikunoshin;
(Uji-shi, JP) |
Correspondence
Address: |
BROWDY AND NEIMARK, P.L.L.C.;624 NINTH STREET, NW
SUITE 300
WASHINGTON
DC
20001-5303
US
|
Assignee: |
Takara Shuzo Co., Ltd.
Kyoto-shi
JP
TAKARA BIO INC.
Kyoto-shi
JP
|
Family ID: |
26397921 |
Appl. No.: |
11/829313 |
Filed: |
July 27, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10657076 |
Sep 9, 2003 |
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11829313 |
Jul 27, 2007 |
|
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09743354 |
Jan 9, 2001 |
6787359 |
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PCT/JP99/03403 |
Jun 25, 1999 |
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10657076 |
Sep 9, 2003 |
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Current U.S.
Class: |
435/405 ;
435/404 |
Current CPC
Class: |
A61K 48/00 20130101;
C12N 2810/80 20130101; C12N 2810/859 20130101; C12N 15/86 20130101;
C12N 2810/851 20130101; C12N 2740/13045 20130101; C12N 2740/13043
20130101; C12N 2810/10 20130101 |
Class at
Publication: |
435/405 ;
435/404 |
International
Class: |
C12N 5/02 20060101
C12N005/02 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 1, 1998 |
JP |
10-186240 |
Mar 4, 1999 |
JP |
11-56915 |
Claims
1. A cell culture medium comprising two functional substances: (1)
a functional substance having an activity of binding to a
retrovirus; and (2) a functional substance which is an antibody
that specifically binds to a target cell.
2. The cell culture medium according to claim 1, wherein the
functional substance having an activity of binding to the
retrovirus is selected from the group consisting of fibronectin,
fibroblast growth factor, collagen type V, polylysine and
DEAE-dextran, as well as fragments thereof and substances having an
equivalent activity of binding to the retrovirus.
3. The cell culture medium according to claim 1, wherein the
functional substance having an activity of binding to the
retrovirus has an activity of binding to the target cell.
4. The cell culture medium according to claim 1, wherein the
functional substance which is an antibody recognizes CD
antigen.
5. The cell culture medium according to claim 1, wherein at least
one of the two functional substances is immobilized on a
substrate.
6. The cell culture medium according to claim 5, wherein said
substrate is a vessel for cell culture or a particulate substrate.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a divisional of application Ser.
No. 10/657,076, filed Sep. 9, 2003, which is a divisional of
application Ser. No. 09/743,354, filed Jan. 9, 2001, which is a 371
national stage of the international application PCT/JP99/03403,
filed Jun. 25, 1999. The entire disclosures of the three
above-identified applications are incorporated herein by
reference.
TECHNICAL FIELD
[0002] The present invention relates to a method that increases the
efficiency of gene transfer into target cells and enables efficient
transduction of the target cells, as well as a series of related
techniques therewith, in the fields of medicine, cell technology,
genetic engineering, developmental technology and the like.
BACKGROUND ART
[0003] Mechanisms of a number of human diseases have been
elucidated. Recombinant DNA techniques and the techniques for
transferring a gene into cells have progressed rapidly. Under these
circumstances, protocols for somatic gene therapies for treating
severe genetic diseases have been recently developed. More
recently, attempts have been made to apply gene therapy not only to
treatment of genetic diseases but also to treatment of viral
infections such as AIDS and cancers.
[0004] In most of the gene therapies which have been examined for
clinical application the humans to date, a gene is transferred into
cells by using a recombinant retrovirus vector. The retrovirus
vector efficiently transfers the foreign gene of interest into
cells and stably integrates the gene into their chromosomal DNA.
Therefore, it is a preferable means of gene transfer, particularly
for gene therapy where long-term gene expression is desired. Such a
vector has been subjected to various modifications so as not to
have a harmful influence on the organism with the transferred gene.
For example, the replication function of the vector is eliminated
such that the vector used for the gene transfer does not replicate
in the cells while repeating unlimited infection (gene
transfer).
[0005] Since such a vector (a replication-deficient retrovirus
vector) cannot autonomously replicate, a retrovirus vector
encapsidated in a virus particle is generally prepared by using
retrovirus-producer cells (packaging cells). The simplest method
for efficiently transferring a gene into target cells comprises
co-culturing the target cells with the retrovirus-producer cells.
However, retrovirus-producer cells may contaminate the gene
transferred target cells which are to be transplanted to a living
body in this method.
[0006] Recently, it was reported that the presence of fibronectin,
a component of the extracellular matrix, or a fragment thereof
increases the efficiency of gene transfer into cells using a
retrovirus (J. Clin. Invest., 93:1451-1457 (1994); Blood,
88:855-862 (1996)). Also, it has been demonstrated that a
fibronectin fragment produced by genetic engineering technique has
similar properties and can be utilized to efficiently transfer a
foreign gene into hematopoietic stem cells (WO 95/26200). It is
suggested that the binding of a heparin-binding region in
fibronectin to a retrovirus is involved in the increase in gene
transfer efficiency due to fibronectin.
[0007] Furthermore, it is disclosed in WO 97/18318 that functional
substances other than fibronectin, such as fibroblast growth
factor, increase gene transfer efficiency. The publication also
discloses that similar increase in gene transfer efficiency is also
observed when a mixture of a functional substance having an
activity of binding to a retrovirus and another functional
substance having an activity of binding to cells is used.
[0008] The gene transfer methods using functional substances enable
an efficient gene transfer without co-cultivating
retrovirus-producer cells and target cells. It is believed that the
increase in gene transfer efficiency by the methods is due to the
increase in the chance of interaction between the retrovirus and
the target cells which are closely co-localized with the aid of the
functional substances.
[0009] In gene transfer using a retrovirus, target cells are
infected with the retrovirus, resulting in gene transfer as
described above. However, the gene transfer efficiency using a
retrovirus is still unsatisfactory for practical clinical
application. Thus, it is desired to further increase infection
efficiency.
[0010] Increased infection efficiency or gene transfer efficiency
may be accomplished by increasing the concentration (titer) of the
retrovirus in the virus suspension (supernatant) used. However,
construction and establishment of virus-producer cells that can
produce high titer viruses usually requires much labor. A
pseudo-type virus vector utilizing an envelope protein from
vesicular stomatitis virus [Proc. Natl. Acad. Sci. USA,
90:8033-8037 (1993)] can be concentrated by centrifugation.
However, since such concentration of the vector can be used only
for this particular vector, it can not be widely used.
[0011] Additionally, specific infection of target cells with a
retrovirus in gene transfer may achieve high gene transfer
efficiency even if the purity of target cells is low. However, no
convenient and efficient method is known in the current state of
the art.
OBJECTS OF INVENTION
[0012] In view of the circumstances described above, the main
object of the present invention is to provide an improved method
for transferring a gene into target cells using a retrovirus, in
which the gene transfer efficiency is increased and the target
cells are efficiently transduced.
[0013] Hereinafter, other objects and advantages of the present
invention will be explained in detail with reference to the
attached drawings.
SUMMARY OF INVENTION
[0014] The present inventors have found that gene transfer
efficiency is increased by contacting recombinant retrovirus with a
functional substance having an activity of binding to a retrovirus,
and being immobilized on a substrate with a retrovirus and then
washing the substrate prior to infecting the target cells.
[0015] The present inventors have also found that a gene can be
transferred specifically for target cells of interest and/or
efficiently by infecting the target cells with a recombinant
retrovirus in the presence of a functional substance, such as an
antibody which specifically binds to the target cells, laminin, a
sugar chain derived from laminin or a high mannose type sugar
chain.
[0016] The present inventors have further found that gene transfer
efficiency can be increased by appropriately pre-treating target
cells before subjecting them to gene transfer.
[0017] Furthermore, the present inventors have found that the
effect on gene transfer of a functional substance having an
activity of binding to a virus can be improved by chemically
modifying the functional substance to increase its basicity.
[0018] The present invention is completed based on these new
findings by the present inventors.
[0019] Thus, the first aspect of the present invention is a method
for transferring a gene into target cells using a retrovirus,
characterized in that the method comprises:
[0020] (1) contacting a solution containing a recombinant
retrovirus with a functional substance having an activity of
binding to the retrovirus and being immobilized on a substrate;
[0021] (2) washing the substrate to which the recombinant
retrovirus is bound; and
[0022] (3) contacting and incubating the substrate to which the
recombinant retrovirus is bound with target cells.
[0023] Without limitation, step (1) above is carried out, for
example, for 1 hour or longer, preferably for 3 hours or longer. In
addition, the frequency of contact between the recombinant
retrovirus and the functional substance having an activity of
binding to the retrovirus may be physically increased.
[0024] Examples of the functional substances having an activity of
binding to the retrovirus which can be used in the present
invention include, but are not limited to, fibronectin, fibroblast
growth factor, collagen type V, polylysine and DEAE-dextran, as
well as fragments thereof and substances having an equivalent
activity of binding to the retrovirus thereto. The functional
substance may have an activity of binding to target cells.
Alternatively, the functional substance may be used in combination
with another functional substance having an activity of binding to
the target cells. Examples of the functional substances having an
activity of binding to the target cells which can be used include,
but are not limited to, cell-adhesive proteins, hormones,
cytokines, antibodies, sugar chains, carbohydrates and
metabolites.
[0025] For example, a culture supernatant of retrovirus-producer
cells can be used as a source of retrovirus for gene transfer in
the present invention. The culture supernatant may be obtained in
the presence of a substance that enhances retrovirus production
such as sodium butyrate.
[0026] The second aspect of the present invention is a method for
transferring a gene into target cells using a recombinant
retrovirus, characterized in that the method comprises infecting
target cells with a recombinant retrovirus in the presence of two
functional substances:
[0027] (1) a functional substance having an activity of binding to
the retrovirus; and
[0028] (2) an antibody which specifically binds to the target
cells.
[0029] Examples of antibodies which specifically bind to the target
cells used in the present invention include, but are not limited
to, an antibody that recognizes a biological substance on the
surface of the target cells.
[0030] The third aspect of the present invention is a method for
transferring a gene into target cells using a recombinant
retrovirus, characterized in that the method comprises infecting
target cells with a recombinant retrovirus in the presence of two
functional substances:
[0031] (1) a functional substance having an activity of binding to
the retrovirus; and
[0032] (2) laminin, a laminin fragment, a sugar chain derived from
laminin or a high mannose type sugar chain.
[0033] Examples of the functional substances having an activity of
binding to the retrovirus which can be used in the second and third
aspects of the present invention include, but are not limited to,
fibronectin, fibroblast growth factor, collagen type V, polylysine
and DEAE-dextran, as well as fragments thereof and substances
having an equivalent activity of binding to the retrovirus. The
functional substance may have an activity of binding to target
cells. Furthermore, the functional substance may be used as being
immobilized on an appropriate substrate.
[0034] The fourth aspect of the present invention is a method for
transferring a gene into target cells using a recombinant
retrovirus, characterized in that the method comprises culturing
target cells in a medium that contains Fe at a low concentration
before the target cells are contacted with the recombinant
retrovirus. Examples of culture media which can be used in the
present invention include, but are not limited to, a medium that
contains deferoxamine. Preferably, the method is carried out in the
presence of a functional substance.
[0035] The fifth aspect of the present invention relates to a
method for increasing an activity of a peptide or a protein for
binding to a retrovirus, characterized in that the method comprises
chemically modifying the peptide or the protein. Examples of
chemical modifications include, but are not limited to, activation
of an amino acid residue in the peptide or the protein and
introduction of a basic residue. For example, the activation of an
amino acid residue is preferably carried out by treating the
peptide or the protein with a water-soluble carbodiimide or with a
water-soluble carbodiimide and a diamino compound, without
limitation. The chemically modified peptide or protein obtained by
the method preferably can be used for gene transfer into target
cells using a retrovirus.
BRIEF DESCRIPTION OF DRAWINGS
[0036] FIG. 1 illustrates a structure of a high mannose type sugar
chain containing nine mannose residues in the molecule.
[0037] FIG. 2 is a graph that shows the gene transfer efficiency
(%) achieved by using chemically modified CH-296 in Example 3.
[0038] FIG. 3 is a graph that shows the relationship between
relative gene transfer efficiency (%) and contact/binding time in a
study on the effect of removing viral infection-inhibitory
substances in Example 13.
[0039] FIG. 4 is a graph which shows the relationship between
relative gene transfer efficiency (%) and respective virus-binding
procedures in a study on the effect of binding retroviruses to
functional substances utilizing the centrifugation method in
Example 15.
[0040] FIG. 5 is a graph that shows the gene transfer efficiency
(%) achieved by using the centrifugation method or the
centrifugation-infection method in Example 15.
DETAILED DESCRIPTION OF THE INVENTION
[0041] A recombinant retrovirus vector is usually used in the gene
transfer method of the present invention. In particular, a
replication-deficient recombinant retrovirus vector is preferably
used. The ability of such a vector to replicate is eliminated so
that it cannot autonomously replicate in infected cells and,
therefore, the vector is non-pathogenic. The vector can invade a
host cell, such as a vertebrate cell (particularly, a mammalian
cell), and stably integrate a foreign gene inserted within the
vector into the chromosomal DNA.
[0042] In the present invention, the foreign gene to be transferred
into the cells can be inserted into the recombinant retrovirus
vector under the control of an appropriate promoter, for example,
the LTR promoter in the retrovirus vector or a foreign promoter. In
addition, another regulatory element (e.g., an enhancer sequence or
a terminator sequence) which cooperates with the promoter and a
transcription initiation site may also be present in the vector in
order to achieve efficient transcription of the foreign gene. The
foreign gene to be transferred may be a naturally occurring gene or
an artificially prepared gene. Alternatively, the foreign gene may
be one in which DNA molecules of different origin are joined
together by ligation or other means known in the art.
[0043] One can select any gene, where its transfer into cells is
desired, as the foreign gene to be inserted into the retrovirus
vector. For example, a gene encoding an enzyme or a protein
associated with the disease to be treated, an intracellular
antibody (see, for example, WO 94/02610), a growth factor, an
antisense nucleic acid, a ribozyme, a false primer (see, for
example, WO 90/13641) or the like can be used as the foreign
gene.
[0044] The retrovirus vector used in the present invention may
contain a suitable marker gene that enables the selection of gene
transferred cells. For example, a drug-resistance gene that confers
resistance of cells to antibiotics or a reporter gene that makes it
possible to distinguish the gene transferred cells by detecting its
enzymatic activity can be utilized as the marker gene.
[0045] The vectors that can be used in the present invention
include, for example, retrovirus vectors such as MFG vector (ATCC
No. 68754), .alpha.-SGC vector (ATCC No. 68755) and LXSN vector
[BioTechniques, 7:980-990 (1989)]. Retrovirus vectors used in the
Examples hereinbelow, including PM5neo vector [Exp. Hematol.,
23:630-638 (1995)], contain a neomycin phosphotransferase gene as a
marker gene. Thus, cells into which a gene is transferred using the
vector can be confirmed based on their resistance to G418.
[0046] These vectors can be prepared as virus particles into which
the vectors are packaged by using a known packaging cell line such
as PG13 (ATCC CRL-10686), PG13/LNc8 (ATCC CRL-10685), PA317 (ATCC
CRL-9078), GP+E-86 (ATCC CRL-9642), GP+envAm12 (ATCC CRL-9641) and
.phi.CRIP [Proc. Natl. Acad. Sci. USA, 85:6460-6464 (1988)].
[0047] Known media, such as Dulbecco's Modified Eagle's Medium and
Iscoves Modified Dulbecco's Medium, can be used for culturing
virus-producer cells which are produced by transferring a
retrovirus vector into packaging cells, or for culturing target
cells. Such media are commercially available, for example, from
Gibco. Various constituents can be added to these media depending
on the type of target cells used for gene transfer or for other
objects of the invention. For example, serum or various cytokines
can be added to the media in order to promote or suppress the
growth or the differentiation of the target cells. For example,
calf serum (CS) or fetal calf serum (FCS) can be used as the serum.
The cytokine includes interleukins (IL-3, IL-6 etc.),
colony-stimulating factors (G-CSF, GM-CSF etc.), stem cell factor
(SCF), erythropoietin and various cell growth factors. Many of
these cytokines derived from humans are commercially available. One
having the suitable activity for the objects of the invention is
selected from the cytokines. Optionally, the cytokines may be used
as a combination of cytokines.
[0048] A sample containing a recombinant retrovirus such as a
culture supernatant of virus-producer cells is used for the gene
transfer method of the present invention. The method for preparing
the supernatant is not limited to a specific method. For example,
it is known that addition of sodium butyrate during cultivation of
virus-producer cells increases the amount of virus particles
produced in the supernatant [Human Gene Therapy, 6:1195-1202
(1995)]. The thus prepared high-titer virus supernatant can be used
in the gene transfer method of the present invention without any
problems.
[0049] The method of the present invention is characterized in that
target cells are infected with a recombinant retrovirus in the
presence of a functional substance having a retrovirus-binding
site. Gene transferred cells can be efficiently obtained by
infecting cells with a recombinant retrovirus in the presence of an
effective amount of such a functional substance. Furthermore, viral
infection-inhibitory substances in a virus supernatant can be
readily removed by using the functional substance. Additionally,
the presence of a functional substance having an activity of
binding to target cells enables gene transfer with higher
specificity and/or efficiency.
[0050] As used herein, an effective amount is an amount effective
to result in transduction of target cells by gene transfer using a
recombinant retrovirus. A suitable amount is selected depending on
the functional substance to be used and the type of target cells.
The amount can be determined, for example, by measuring the
efficiency of gene transfer by the method as described herein. As
used herein, activities of binding to target cells include not only
an activity of substantially binding to cells but also an activity
of keeping in contact with target cells in a solution. The
activities can be measured based on the contribution to gene
transfer efficiency as described above. In addition, gene transfer
efficiency means the efficiency of transduction.
[0051] The above-mentioned functional substances can be used either
dissolved in a solution or immobilized on an appropriate substrate.
The substrate for immobilizing a functional substance is not
limited to any specific substance. Usually, a vessel for cell
culture or a bead-shaped substrate is used.
[0052] When a functional substance having an activity of binding to
a virus and being immobilized on a substrate is used, the
efficiency of gene transfer can be further increased by using the
steps exemplified below.
[0053] First, a liquid sample (e.g., a virus supernatant)
containing a recombinant retrovirus is contacted with a substrate
on which a functional substance having an activity of binding to a
retrovirus is immobilized. The substrate is washed. The substrate
is then directly contacted with target cells. Alternatively, virus
particles eluted from the substrate by an appropriate means are
added to target cells. Thus, a gene can be efficiently transferred.
The functional substance having an activity of binding to a
recombinant retrovirus may also have an activity of binding to
target cells. Alternatively, a functional substance having an
activity of binding to a recombinant retrovirus and a functional
substance having an activity of binding to target cells may be used
in combination.
[0054] A step of contacting a liquid sample containing a
recombinant retrovirus with a substrate on which a functional
substance having an activity of binding to the recombinant
retrovirus is immobilized is conducted, for example, for 1 hour or
longer, preferably for three hours or longer, without limitation.
Also, other conditions including temperature are not specifically
limited. For example, the step can be conducted at room temperature
or 37.degree. C. Low temperatures around 4.degree. C. may be used
depending on the stability of the virus or the like. The substrate
for immobilizing a functional substance may be appropriately
selected depending on the object of the invention. If a vessel for
cell culture is used, one can start gene transfer only by adding
target cells. For example, phosphate buffered saline or Hanks'
saline, as the liquid medium used for culturing target cells or the
like, can be used for washing the substrate.
[0055] A retrovirus can be more efficiently bound to a functional
substance having an activity of binding to the retrovirus by
physically increasing the frequency of contact between the
retrovirus and the functional substance. Examples of such physical
means include, but are not limited to, shaking, filtration and
centrifugal force. The use of centrifugal force is specifically
exemplified by a method in which a liquid sample containing a
retrovirus is added to a centrifugation tube in which a functional
substance having an activity of binding to the retrovirus is
immobilized at the bottom and the centrifugation tube is then
centrifuged. The retrovirus is precipitated onto the bottom of the
centrifugation tube by centrifugal force during centrifugation.
Accordingly, the frequency of contact between the retrovirus and
the functional substance having an activity of binding to the
retrovirus is increased, resulting in an increase in the frequency
of binding. The above-mentioned method does not put the cells under
a physical stress like the method in which viruses are precipitated
onto cells by centrifugal force for purpose of infection (WO
95/10619). Thus, the method of the present invention results in
higher gene transfer efficiency.
[0056] Gene transfer can be conducted after removing a substance
contained in a retrovirus sample, whose presence is not beneficial
for gene transfer, by the procedure described above. For example,
substances removed by the method of the present invention include a
retroviral infection-inhibitory substance derived from packaging
cells contained in a virus supernatant [Human Gene Therapy,
8:1459-1467 (1997); J. Virol., 70:6468-6473 (1996)], substances
added during culturing retrovirus-producer cells in order to
enhance retrovirus production such as phorbol 12-myristate
13-acetate (TPA) and dexamethasone [Gene Therapy, 2:547-551
(1995)], as well as sodium butyrate as described above.
[0057] Examples of functional substances having an activity of
binding to a retrovirus which can be used in the present invention
include, but are not limited to, heparin-II domain of fibronectin,
fibroblast growth factor, collagen type V, polylysine and
DEAE-dextran, as well as substances functionally equivalent to
these functional substances (e.g., a functional substance having a
heparin-binding site). Furthermore, a mixture of the functional
substances, a polypeptide containing the functional substance, a
polymer of the functional substance, a derivative of the functional
substance and the like can be used.
[0058] The functional substance's activity of binding to a virus
can be enhanced by chemically modifying it. Examples of chemical
modifications include activation of an amino acid residue in the
functional substance used and introduction of a basic residue into
the substance. For example, the activity of binding to a retrovirus
can be increased by modifying a free carboxyl group in a functional
substance consisting of a peptide or a protein with a water-soluble
carbodiimide such as 1-ethyl-3-dimethylaminopropylcarbodiimide
hydrochloride to activate the carboxyl group. Furthermore, the
activity of binding to a retrovirus can be increase by use the thus
activated carboxyl group to introduce a basic residue, such as an
amino group into the functional substance.
[0059] Examples of the functional substance having an activity of
binding to target cells used in the present invention include, but
are not limited to, a substance that has a ligand that binds to the
target cells. The ligands include cell-adhesive proteins, hormones
or cytokines, antibodies against cell surface antigens,
polysaccharides, glycoproteins, glycolipids, sugar chains derived
from glycoproteins or glycolipids, and metabolites of the target
cells. Furthermore, a polypeptide containing the functional
substance, a polymer of the functional substance, a derivative of
the functional substance, a functional equivalent of the functional
substance or the like can be used.
[0060] An antibody that specifically binds to target cells is
particularly useful for specifically and efficiently transferring a
gene into specific cells. The antibody which can be used in the
present invention is not limited to any specific antibody. An
antibody against an antigen expressed on target cells into which a
gene is to be transferred can be appropriately selected for use.
Such an antibody can be produced according to known methods.
Alternatively, many current commercially available antibodies can
also be used. The antibody may be a polyclonal antibody or a
monoclonal antibody as long as it has desired properties such as
cell specificity. Additionally, an antibody or a derivative of an
antibody modified using known techniques such as a humanized
antibody, a Fab fragment or a single-chain antibody can also be
used.
[0061] Expression of leukocyte antigens (also known as CD antigens)
on various cells has been studied in detail. Thus, a gene can be
transferred into target cells with high specificity by selecting an
antibody that recognizes a CD antigen expressed on the target cells
of interest and using it in the gene transfer method of the present
invention. For example, gene transfer can be directed to helper T
cells by using an anti-CD4 antibody, or to hematopoietic stem cells
by using an anti-CD34 antibody.
[0062] Furthermore, a glycoprotein, laminin, can be used as a
functional substance having an activity of binding to target cells
to efficiently transfer a gene into various target cells such as
hematopoietic cells. The laminin which can be used in the present
invention may be derived from mouse or human, or it may be a
fragment thereof as long as it has an activity of binding to target
cells. As described in the examples below, the sugar chain of
laminin plays an important role in gene transfer using laminin.
Therefore, a sugar chain released from laminin according to a known
method can also be used in the method of the present invention.
Furthermore, a glycoprotein having a high mannose type N-linked
sugar chain like laminin, or a sugar chain released therefrom or
chemically synthesized, can also be used in the present invention.
Additionally, a substance such as a protein or the like having the
above-mentioned sugar chain being attached thereto can be used. For
example, a functional substance having an activity of binding to a
retrovirus and having the sugar chain attached thereto can
preferably be used for gene transfer.
[0063] The above-mentioned high mannose type sugar chain is not
limited to a specific chain as long as it has 1 to 20 mannose
residues in the molecule. A sugar chain having a mannose residue at
its non-reducing end is preferably used in the method of the
present invention. The sugar chain can be used by being attached to
another appropriate molecule such as a biological molecule (e.g., a
monosaccharide, an oligosaccharide, a polysaccharide, an amino
acid, a peptide, a protein or a lipid) or an artificial substance
such as a synthetic macromolecule.
[0064] Representative high mannose type sugar chains derived from
organisms are exemplified by those having a structure represented
by (Mannose).sub.n-(GlucNAc).sub.2 [Protein, Nucleic Acid and
Enzyme, 43:2631-2639 (1998)]. For example,
(Mannose).sub.9-(GlucNAc).sub.2, a sugar chain which has the
structure as described above and contains nine mannose residues in
the molecule, can preferably be used in the gene transfer method of
the present invention, without limitation (the structure of this
sugar chain is shown in FIG. 1).
[0065] The functional substance as described above can be obtained
from naturally occurring substances, an artificial preparation (for
example, by recombinant DNA techniques or chemical synthesis
techniques), or a preparation that combines a naturally occurring
substance and an artificially prepared substance. In addition, a
mixture of a functional substance that has a retrovirus-binding
site and another functional substance that has a target
cell-binding site can be used for the gene transfer using the
functional substances as described in WO 97/18318. Alternatively, a
functional substance that has a retrovirus-binding site and a
target cell-binding site in a single molecule can be used.
Functional substances substantially free of other proteins
naturally associated the functional substances are used.
Additionally, the functional substance or a combination of the
functional substances can be combined with a medium used for
culturing target cells, cell growth factor and the like to produce
a kit for gene transfer.
[0066] Fibronectin or a fragment thereof used in the method of the
present invention can be prepared in a substantially pure form from
naturally occurring materials according to methods described, for
example, in J. Biol. Chem., 256:7277 (1981); J. Cell. Biol.,
102:449 (1986); or J. Cell. Biol., 105:489 (1987). The fibronectin
or the fragment thereof can be prepared using recombinant DNA
techniques as described in U.S. Pat. No. 5,198,423. Specifically, a
fibronectin fragment containing the heparin-II domain, which is a
retrovirus-binding site, such as the recombinant polypeptides
including CH-296, H-271, H-296 and CH-271 used in the Examples
below as well as the method for obtaining them are described in
detail in the above-mentioned patent. These fragments can be
obtained by culturing Escherichia coli strains deposited under
accession numbers FERM P-10721 (H-296) (the date of the original
deposit: May 12, 1989), FERM BP-2799 (CH-271) (the date of the
original deposit: May 12, 1989), FERM BP-2800 (CH-296) (the date of
the original deposit: May 12, 1989) and FERM BP-2264 (H-271) (the
date of the original deposit: Jan. 30, 1989) at the National
Institute of Bioscience and Human-Technology, Agency of Industrial
Science and Technology, Ministry of International Trade and
Industry, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken as
described in the patent publication. In addition, fragments that
can be typically derived from these fibronectin fragments can be
prepared by modifying the plasmids harbored in the Escherichia coli
strains above using known recombinant DNA techniques. Among the
fibronectin fragments described above, H-296 the binding region to
VLA-4, CH-271 has the binding region to VLA-5, and CH-296 has both
[Nature Medicine, 2:876-882 (1996)].
[0067] Gene transferred cells can be efficiently obtained by
infecting target cells with a recombinant retrovirus in the
presence of the functional substance. The infection with the
recombinant retrovirus can be carried out according to a
conventional method, for example, by incubating at 37.degree. C. in
5% CO.sub.2. These conditions and the incubation time may be
suitably changed depending on the target cells or the objects of
the invention.
[0068] Target cells are not infected with a retrovirus when they
are in the G.sub.0 phase. Therefore, it is preferable to lead the
cells into the cell cycle by pre-stimulating them. For this
purpose, the target cells are cultured in the presence of a growth
factor suitable for the target cells prior to the infection of the
cells with the retrovirus. For example, various cytokines such as
interleukin-3, interleukin-6 and stem cell factor are used to
pre-stimulate bone marrow cells or hematopoietic stem cells for
gene transfer.
[0069] It is known that receptors on the surface of cells are
involved in infection of cells with retroviruses. Basic amino acid
transporter and phosphate transporter are known to function as
receptors for ecotropic viruses and amphotropic viruses,
respectively [Proc. Natl. Acad. Sci. USA, 93:11407-11413
(1996).quadrature.. It is possible to make target cells susceptible
to viral infection by pre-treating the cells in a medium in which
concentrations of basic amino acids or phosphates, or salts or
precursors thereof, are reduced in order to activate the expression
or metabolic turnover of the transporters.
[0070] Surprisingly, the present inventors have found that
activation of the transferring receptor, whose involvement in viral
infection was not previously known, also increases the efficiency
of retroviral infection or gene transfer efficiency. The
transferring receptor can be activated, without limitation, by
treating target cells in a medium containing a limited
concentration of Fe. For example, a medium in which Fe is chelated
by adding deferoxamine can be used.
[0071] Preferably, gene transfer using transferring activation is
also carried out in the presence of the functional substance as
described above.
[0072] Examples of cells which can be used as the target for gene
transfer by the method of the present invention include, but are
not limited to, stem cells, hematopoietic cells, non-adhesive
low-density mononuclear cells, adhesive cells, bone marrow cells,
hematopoietic stem cells, peripheral blood stem cells, umbilical
cord blood cells, fetal hematopoietic stem cells, embryogenic stem
cells, embryonic cells, primordial germ cells, oocytes, oogonia,
ova, spermatocytes, sperms, CD34+ cells, c-kit+ cells, pluripotent
hematopoietic progenitor cells, unipotent hematopoietic progenitor
cells, erythroid precursor cells, lymphoid mother cells, mature
blood cells, lymphocytes, B cells, T cells, fibroblasts,
neuroblasts, neurocytes, endothelial cells, vascular endothelial
cells, hepatocytes, myoblasts, skeletal muscle cells, smooth muscle
cells, cancer cells, myeloma cells, leukemia cells, and so on. The
method of the present invention is preferably used for
hematopoietic cells which are available from blood and bone marrow
because these cells are relatively easy to obtain and because the
techniques for culturing and maintaining them are established. In
particular, if long-term expression of the transferred gene is
intended, then blood progenitor cells such as hematopoietic stem
cells, CD34-positive cells, c-kit-positive cells and pluripotent
hematopoietic progenitor cells are suitable as target cells.
[0073] For example, gene therapy using hematopoietic stem cells as
target cells can be carried out by the following procedure.
[0074] First, a material containing hematopoietic stem cells such
as bone marrow tissue, peripheral blood and umbilical cord blood is
collected from a donor. Such a material can be directly used in the
gene transfer procedure. However, mononuclear cell fractions
containing hematopoietic stem cells are usually prepared by means
of density-gradient centrifugation and the like, or hematopoietic
stem cells are further purified by utilizing cell surface marker
molecules such as CD34 and/or c-kit. The material containing the
hematopoietic stem cells is infected with a recombinant retrovirus
vector, into which a gene of interest is inserted according to the
method of the present invention, after being pre-stimulated by
using a suitable cell growth factor, if necessary. The gene
transferred cells thus obtained can be transplanted into a
recipient, for example, by intravenous administration. Although the
recipient is preferably the donor itself, allogenic transplantation
can also be carried out. For example, if the umbilical cord blood
is used as the material, allogenic transplantation is
performed.
[0075] Some gene therapies using hematopoietic stem cells as target
cells are for complementing a deficient or abnormal gene in a
patient (e.g., gene therapy for ADA deficiency or Gaucher's
disease). In addition, a drug resistance gene may be transferred
into hematopoietic stem cells in order to alleviate the damage due
to the chemotherapeutic agents used for, e.g., the treatment of
cancer or leukemia.
[0076] Tumor vaccination therapy is investigated as a gene therapy
for cancer. In such a therapy, a gene for a cytokine is transferred
into cancer cells, the cancer cells are deprived of the ability to
proliferate, and the cells are then returned to the body of the
patient to enhance tumor immunity [Human Gene Therapy, 5:153-164
(1994)]. In addition, attempts are made to treat AIDS using gene
therapy. In this case, the following procedure is considered. In
the procedure, a gene encoding a nucleic acid molecule (e.g., an
antisense nucleic acid or a ribozyme) which interferes with the
replication or the gene expression of HIV (human immunodeficiency
virus) is transferred into T cells infected with HIV, the causal
agent of AIDS [e.g., J. Virol., 69:4045-4052 (1995)].
[0077] As described above in detail, a gene can be transferred with
high efficiency and with high specificity for target cells by using
the present invention. Furthermore, the method of the present
invention does not require any specialized equipment or instrument
and is effective for various retrovirus vectors and target
cells.
EXAMPLES
[0078] The following Examples illustrate the present invention in
more detail, but are not to be construed to limit the scope
thereof.
Example 1
Preparation of Polypeptides Derived from Fibronectin
[0079] A polypeptide derived from human fibronectin, H-271, was
prepared from Escherichia coli HB101/pHD101 (FERM BP-2264) carrying
a recombinant plasmid pHD101 which contains a DNA encoding the
polypeptide according to the method as described in U.S. Pat. No.
5,198,423.
[0080] A polypeptide derived from human fibronectin, H-296, was
prepared from Escherichia coli HB101/pHD102 (FERM P-10721) carrying
a recombinant plasmid pHD102 which contains a DNA encoding the
polypeptide according to the method as described in the
above-mentioned publication.
[0081] A polypeptide CH-271 was prepared as follows.
[0082] Briefly, Escherichia coli HB101/pCH101 (FERM BP-2799) was
cultured according to the method as described in the
above-mentioned publication. CH-271 was obtained from the
culture.
[0083] A polypeptide CH-296 was prepared as follows.
[0084] Briefly, Escherichia coli HB101/pCH102 (FERM BP-2800) was
cultured according to the method as described in the
above-mentioned publication. CH-296 was obtained from the
culture.
[0085] A polypeptide C-274 was prepared as follows.
[0086] Briefly, Escherichia coli JM109/pTF7221 (FERM BP-1915) was
cultured according to the method as described in U.S. Pat. No.
5,102,988. C-274 was obtained from the culture.
[0087] A polypeptide having an activity of binding to a retrovirus
derived from collagen type V, ColV, was prepared according to the
method as described in WO 97/18318.
Example 2
Construction of Retrovirus Vector and Preparation of Retrovirus
Supernatant
[0088] A retrovirus plasmid, PM5neo vector, which contains a
neomycin phosphotransferase gene [Exp. Hematol., 23:630-638 (1995)]
was introduced into GP+E-86 cells (ATCC CRL-9642). The cells were
cultured in Dulbecco's Modified Eagle's Medium (DMEM; Bio
Whittaker) containing 10% fetal calf serum (FCS; Gibco), 50
units/ml of penicillin and 50 .mu.g/ml of streptomycin (both from
Gibco). All of the DMEMs used in the procedure hereinbelow contain
the above-mentioned antibiotics. A supernatant containing PM5neo
virus was prepared by adding 4 ml of DMEM containing 10% FCS to a
plate (a 10-cm gelatin-coated dish for cell culture, Iwaki Glass)
in which the above-mentioned producer cells had been grown to
semi-confluence, culturing overnight and then collecting the
supernatant. The thus collected culture supernatant was filtered
through a 0.45-micron filter (Millipore) to prepare a virus
supernatant stock, which was stored at -80.degree. C. until
use.
[0089] Virus supernatants were prepared from the following cells
according to the procedure described above. Ecotropic packaging
BOSC23 cells [Proc. Natl. Acad. Sci. USA, 90:8392-8396 (1993)],
into which a retrovirus plasmid pLEIN (Clontech; which contains a
neomycin phosphotransferase gene and an enhanced green fluorescent
protein (EGFP) gene) had been introduced; and amphotropic packaging
.phi.CRIP cells [Proc. Natl. Acad. Sci. USA, 85:6460-6464 (1988)].
Hereinafter, a virus prepared from BOSC23 cells is referred to as
Eco-EGFP, and a virus prepared from .phi.CRIP cells is referred to
as Ampho-EGFP, respectively.
[0090] Furthermore, a virus supernatant was prepared from
GP+EnvAm12 cells (ATCC CRL-9641) carrying a retrovirus plasmid,
TKNeo vector [J. Exp. Med., 178:529-536 (1993)] (which contains a
neomycin phosphotransferase gene) according to the procedure as
described above. DMEM containing 10% calf serum (CS; Gibco) in
place of FCS was used.
[0091] The titer of the virus supernatant was measured according to
a standard method [J. Virol., 62:1120-1124 (1988)] in which the
efficiency of transferring a neomycin phosphotransferase gene into
NIH/3T3 cells (ATCC CRL-1658) is used as an index. The number of
infectious particles contained in 1 ml of the supernatant (cfu/ml)
was calculated. The amount of the virus supernatant to be added in
the experiments hereinbelow was determined based on the calculated
value, i.e., the titer of the supernatant.
Example 3
Preparation of the Functional Substance Having Activity of Binding
to Retrovirus and Measurement of Activity thereof
[0092] 50 .mu.l of 80 .mu.g/ml solution of H-271, H-296, C-274,
CH-271, CH-296, ColV, human basic fibroblast growth factor (bFGF;
Progen), tenascin (Gibco) or epidermal growth factor (EGF; Takara
Shuzo), or 50 .mu.l of 2% bovine serum albumin (BSA, Sigma) was
added to each well of a 96-well non-treated microplate for cell
culture (Falcon). The plate was allowed to stand at 4.degree. C.
overnight and then washed twice with phosphate buffered saline
(PBS; Roman Kogyo). Alternatively, after the plate was treated as
described above, 0.1 ml of 4 mg/ml solution of
1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (Sigma) in
sterile pure water was dispensed to each well. The reaction was
allowed to proceed at 37.degree. C. for 2 hours. The plate was
washed extensively with pure water to prepare a
carbodiimide-treated plate. These plates were stored at 4.degree.
C. until the viral infection experiments were conducted.
[0093] 10.sup.4 mouse leukemia L1210 cells (ATCC CCL-219), which
had been grown in RPMI 1640 medium (Bio Whittaker) supplemented
with 10% FCS, 50 units/ml penicillin and 50 .mu.g/ml streptomycin,
and 50 .mu.l of PM5neo virus supernatant (10.sup.4 cfu/ml) were
added to the well of the microplate. After the plate was incubated
for 24 hours, the medium was changed to the same medium containing
G418 (Gibco) at a final concentration of 0.75 mg/ml, and the plate
was then incubated for additional 48 hours. G418-resistance cells
were assessed according to the method described in S. Kim et al.
[Gene Therapy, 3:1018-1020 (1996)] with a partial modification in
which the color developed using Premix WST-1 reagent (Takara Shuzo)
was measured as absorbance at 450 nm. After incubation, 10
.mu.l/100 .mu.l culture of the WST-1 reagent was added to the well,
the plate was incubated at 37.degree. C. for additional 4 hours.
Absorbance at 450 nm and 650 nm was then measured using a
microplate reader, and the difference (450 nm-650 nm) was
calculated. The value obtained using a plate coated with 2% BSA
without carbodiimide treatment was defined as background. The
results from three rounds of studies are summarized in Table 1.
TABLE-US-00001 TABLE 1 Functional Treated with substance Untreated
carbodiimide Experiment 1 BSA 0.000 .+-. 0.011 Not done CH-271
2.099 .+-. 0.010 2.814 .+-. 0.079 Experiment 2 BSA 0.000 .+-. 0.007
0.224 .+-. 0.031 H-271 0.777 .+-. 0.016 0.994 .+-. 0.029 H-296
0.474 .+-. 0.014 0.666 .+-. 0.021 C-274 -0.068 .+-. 0.017 0.100
.+-. 0.033 CH-271 0.382 .+-. 0.017 0.425 .+-. 0.019 CH-296 0.363
.+-. 0.023 0.460 .+-. 0.007 ColV 0.644 .+-. 0.006 0.847 .+-. 0.033
bFGF 0.425 .+-. 0.014 0.580 .+-. 0.046 Tenascin 0.060 .+-. 0.021
0.323 .+-. 0.037 EGF 0.030 .+-. 0.021 0.077 .+-. 0.038 (Mean .+-.
standard deviation)
[0094] As shown in Table 1, an increase in gene transfer efficiency
was observed for known functional substances having an activity of
binding to a virus, i.e., H-271, H-296, CH-271, CH-296, ColV and
bFGF. Furthermore, the appearance of G418-resistant cells increased
when C-274, tenascin, EGF and BSA, which do not have an activity of
binding to a virus, were used and the carbodiimide treatment was
carried out.
[0095] Next, CH-296 was used as a functional substance to carry out
experiments as follows.
[0096] 0.5 ml of 40 .mu.g/ml CH-296 was added to each well of a
24-well non-treated microplate for cell culture (Falcon). The plate
was incubated at 4.degree. C. overnight and then washed with PBS
(pH 5.8). 625 .mu.l of 10 mg/ml solution of
1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (Sigma) in
PBS (pH 5.8) containing ethylenediamine
[NH.sub.2(CH.sub.2).sub.2NH.sub.2; Nacalai Tesque],
trimethylenediamine [NH.sub.2(CH.sub.2).sub.3NH.sub.2; Nacalai
Tesque] or putrescine [NH.sub.2(CH.sub.2).sub.4NH.sub.2; Nacalai
Tesque] at a varying concentration was added to each well. The
plate was incubated at 37.degree. C. for 2 hours. An amino group
was introduced to the carboxyl group in the CH-296 molecule through
the mediation of carbodiimide in this procedure. The plate was
washed three times with PBS, and then blocked with 2% glycine/PBS
followed by 2% BSA/PBS.
[0097] GP+E86 cells, into which a retrovirus vector plasmid pLEIN
had been introduced, were cultured in DMEM containing 10% CS. A
supernatant was then collected from the culture. 0.5 ml of a virus
supernatant prepared by diluting the supernatant to make the
concentration to 1.times.10.sup.5 cfu/ml was added to each well of
the plate. The plate was incubated for 4 hours. 1.times.10.sup.4
NIH/3T3 cells were further added to the well. The plate was
incubated for 2 days. After incubation, the cells were collected by
using a cell detachment buffer (Bio Whittaker) and washed.
EGFP-expressing cells were analyzed by flow cytometry using
FACSVantage.TM. (Becton Dickinson) at an excitation wavelength of
488 nm and an emission wavelength of 515-545 nm. The binding
ability of the virus to the plate was expressed by the efficiency
of gene transfer into cells. The results are shown in FIG. 2.
[0098] As shown FIG. 2, the binding ability of the virus increased
as the concentration of the diamino compound used for introducing
an amino group increased. About a two-fold increase in the binding
ability of the virus was observed when putrescine,
trimethylenediamine or ethylenediamine was used in the reaction at
a concentration of 2 mM as compared with the binding ability
observed using untreated CH-296.
Example 4
Effect of Enhancing Gene Transfer Efficiency of Laminin
[0099] Mouse laminin (Gibco) or human laminin (Takara Shuzo) was
used in combination with a functional substance having an activity
of binding to a virus to carry out gene transfer experiment. A
24-well non-treated microplate for cell culture (Falcon) used in
the experiment was coated with these functional substances
according to the following two methods.
[0100] The cocktail method: A mixture of two functional substances
is added to the plate. The plate is allowed to stand at 4.degree.
C. overnight. The plate is blocked with 2% BSA at 37.degree. C. for
20 minutes and then washed with PBS.
[0101] The pre-coating method: A solution of a functional substance
having an activity of binding to a virus is added to the plate. The
plate is allowed to stand at 4.degree. C. overnight. The solution
is removed. A laminin solution is added to the plate. The plate is
incubated at 37.degree. C. for 2 hours, blocked with 2% BSA, and
then washed with PBS.
[0102] 0.5 ml of the solution of the functional substance was used
to coat each well.
[0103] 10.sup.5 L1210 cells and 0.5 ml of Eco-EGFP virus
supernatant (10.sup.5 cfu/ml) were added to the well. The plate was
incubated for 24 hours. After incubation, the cells were collected
by using a cell detachment buffer (Bio Whittaker) and washed.
EGFP-expressing cells were analyzed by flow cytometry using
FACSVantage.TM. (Becton Dickinson) at an excitation wavelength of
488 nm and an emission wavelength of 515-545 nm. The gene transfer
efficiency (the ratio of EGFP-expressing cells to total cells) was
calculated. The experimental results are shown in Tables 2 to 5.
TABLE-US-00002 TABLE 2 Concentration of laminin added and coating
method Functional No 5 .mu.g/ml 20 .mu.g/ml substance addition Pre-
Pre- 20 .mu.g/ml (80 .mu.g/ml) -- coating coating Cocktail BSA (2%)
1.12 5.20 6.55 6.22 H-271 5.41 11.19 17.52 9.67 H-296 4.83 5.96
5.51 6.95 CH-271 4.00 6.72 13.73 17.34 CH-296 6.48 7.08 6.02
16.77
[0104] Gene transfer efficiency in % is indicated. TABLE-US-00003
TABLE 3 Functional Concentration of laminin added substance No (80
.mu.g/ml) addition 20 .mu.g/ml 40 .mu.g/ml 60 .mu.g/ml BSA (2%)
1.36 5.14 4.74 3.82 CH-271 16.89 32.05 24.45 23.46 CH-296 17.80
18.79 20.44 19.31
[0105] The plate was coated according to the cocktail method. Gene
transfer efficiency in % is indicated. TABLE-US-00004 TABLE 4
Concentration of laminin added Concentration of No CH-296 added
addition 5 .mu.g/ml 10 .mu.g/ml 20 .mu.g/ml No addition 0.69 4.09
6.76 6.89 10 .mu.g/ml 4.67 11.81 9.36 7.01 20 .mu.g/ml 5.16 11.64
10.57 8.49 40 .mu.g/ml 4.41 10.49 11.52 9.11 80 .mu.g/ml 5.11 10.87
11.48 11.10 160 .mu.g/ml 5.19 9.04 11.84 10.88 320 .mu.g/ml Not
done Not done 10.27 10.54
[0106] The plate was coated according to the cocktail method. Gene
transfer efficiency in % is indicated. TABLE-US-00005 TABLE 5
Concentration of laminin added Concentration of No CH-271 added
addition 5 .mu.g/ml 10 .mu.g/ml 20 .mu.g/ml No addition 0.69 4.09
6.76 6.89 10 .mu.g/ml 4.61 7.16 6.28 6.34 20 .mu.g/ml 4.71 12.98
8.98 5.99 40 .mu.g/ml 3.64 17.32 14.50 8.78 80 .mu.g/ml 3.60 18.30
14.76 9.15 160 .mu.g/ml 3.52 16.34 17.08 12.67
The plate was coated according to the cocktail method. Gene
transfer efficiency in % is indicated.
[0107] As shown in Tables 2 and 3, it was demonstrated that a gene
was transferred into target cells very efficiently, regardless of
the immobilization method, when mouse or human laminin was used in
combination with a functional substance having an activity of
binding to a virus for gene transfer using a retrovirus. The gene
transfer efficiency using a plate coated with CH-296 or CH-271 and
laminin according to the cocktail method was examined. As shown in
Tables 4 and 5, it revealed that the optimal ratios were 8:1 (e.g.,
80 .mu.g/ml: 10 .mu.g/ml) for the combination of CH-296/mouse
laminin, and 16:1 (e.g., 80 .mu.g/ml: 5 .mu.g/ml) for CH-271/mouse
laminin, respectively. The gene transfer efficiency increased
2.6-fold and 5.1-fold for CH-296 and CH-271, respectively, as
compared with the efficiency of gene transfer without the addition
of laminin.
Example 5
Gene Transfer into Mouse C-Kit-Positive Bone Marrow Cells Using
Laminin
[0108] Mouse c-kit-positive bone marrow cells were prepared as
follows. Bone marrow cells collected from a femur of a 6-8 weeks
old C3H/He female mouse (Japan SLC) were subjected to
density-gradient centrifugation using Ficoll-Hypaque (1.0875 g/ml,
Pharmacia) to prepare a fraction containing low-density mononuclear
cells. The cells were washed with PBS, erythrocytes were lysed
using Ery-Lysis buffer (155 mM NH.sub.4Cl, 10 mM KHCO.sub.3, 0.1 mM
EDTA, pH 7.4), and the cells were washed again with PBS. 1
.mu.g/10.sup.7 cells of an anti-mouse CD117 antibody (Pharmingen)
was added to the resulting bone marrow cells. The mixture was
reacted on ice for 30 minutes. The cells were washed with PBS
containing 5 mM EDTA and 0.5% BSA, and then suspended in the same
buffer. 20 .mu.l/10.sup.7 cells of a secondary antibody conjugated
with a microbead (Miltenyi Biotec) was added to the cells. The
mixture was reacted at 4.degree. C. for 30 minutes. The cells were
washed with and resuspended in the above-mentioned buffer. Cells
bound to the microbeads were collected using MACS system (Miltenyi
Biotec) to obtain c-kit-positive cells.
[0109] Prior to viral infection, the mouse c-kit-positive bone
marrow cells were pre-stimulated in accordance with the method of
Luskey et al. [Blood, 80:396-402 (1992)]. Briefly, cells were
cultured in .alpha.-MEM (Bio Whittaker) containing 20% FCS, 20
ng/ml of recombinant mouse interleukin-3 (Genzyme), 50 ng/ml of
recombinant human interleukin-6 (Genzyme), 100 ng/ml of recombinant
mouse stem cell factor (Genzyme), 50 units/ml of penicillin and 50
.mu.g/ml of streptomycin 37.degree. C. for 2 days in the presence
of 5% CO.sub.2.
[0110] A 24-well non-treated microplate for cell culture was coated
according to the cocktail method using a mixture containing mouse
laminin at a varying concentration and 80 .mu.g/ml of CH-271. The
plate was blocked with 2% BSA for 30 minutes, and then washed with
PBS. A control plate was prepared using 2% BSA in place of CH-271.
105 c-kit-positive bone marrow cells and 0.5 ml Eco-EGFP virus
supernatant (105 cfu/ml) were added to each well of the microplate
for viral infection. After incubation for 48 hours, 0.5 ml of fresh
medium was added to the well, and the plate was incubated for
additional 24 hours. After incubation, the cells were collected by
using a cell detachment buffer and washed. The gene transfer
efficiency was calculated as described in Example 4. The results
from two rounds of experiments are shown in Tables 6 and 7.
TABLE-US-00006 TABLE 6 Concentration of laminin added No addition
10 .mu.g/ml 20 .mu.g/ml BSA 0.18 0.25 0.16 CH-271 0.69 3.93
2.64
[0111] Gene transfer efficiency in % is indicated. TABLE-US-00007
TABLE 7 Concentration of laminin added No addition 2.5 .mu.g/ml 5
.mu.g/ml 10 .mu.g/ml BSA 1.37 1.80 2.63 5.38 CH-271 9.95 16.12
15.28 17.00
Gene transfer efficiency in % is indicated.
[0112] As shown in Tables 6 and 7, it was demonstrated that a very
strong effect of enhancing gene transfer efficiency was also
observed when c-kit-positive bone marrow cells were infected with a
retrovirus in a plate coated with mouse laminin and a functional
substance having an activity of binding to a virus, CH-271,
according to the cocktail method. The efficiency of gene transfer
using CH-271 in combination with laminin increase 5.7-fold at the
most as compared with that using CH-271 alone.
[0113] Furthermore, the same procedure as that described above was
carried out using Eco-EGFP virus supernatant at a titer of 10.sup.7
cfu/ml. The mean gene transfer efficiency from three rounds of
experiments is shown in Table 8. It was also demonstrated in this
case that the efficiency of gene transfer using a functional
substance having an activity of binding to a retrovirus was
increased by using laminin in combination. TABLE-US-00008 TABLE 8
Concentration of laminin added No addition 2 .mu.g/ml 4 .mu.g/ml 6
.mu.g/ml BSA 5.88 11.77 19.33 27.09 H-271 25.12 53.39 55.65 56.45
CH-271 43.06 66.87 73.67 77.76 CH-296 76.84 81.57 83.30 85.48
Gene transfer efficiency in % is indicated.
Example 6
Gene Transfer into CD3-Positive T Cells Derived from Mouse Spleen
Cells using Laminin
[0114] CD3-positive T cells derived from mouse spleen cells were
prepared as follows. Cells were collected from a spleen of a 6-8
weeks old C3H/He female mouse. The cells were passed through a
100-.mu.m mesh (Falcon) to remove residuals. The resulting cells
were washed with Hanks' balanced salt solution (HBSS, Bio
Whittaker) containing 10% FCS, erythrocytes were lysed using
Ery-Lysis buffer, and the cells were washed again with HESS. The
resulting cells were passed through a 30-.mu.m mesh (Miltenyi
Biotec) to remove residuals and then purified using a column for
concentrating CD3-positive T cells (R&D Systems). Mouse
CD3-positive T cells used for viral infection experiments were
pre-stimulated as follows. The cells were cultured for
pre-stimulation in a Petri dish onto which an anti-mouse CD3
antibody and an anti-mouse CD28 antibody (both at 1 .mu.g/ml,
Pharmingen) had been immobilized. The Petri dish contained RPMI
1640 medium (Bio Whittaker) supplemented with 10% FCS, 50 units/ml
of penicillin and 50 .mu.g/ml of streptomycin. The cells were
cultured at 37.degree. C. for 2 days in the presence of 5%
CO.sub.2.
[0115] A 24-well microplate was coated using a mixture containing
20 .mu.g/ml of mouse laminin and 80 .mu.g/ml of CH-296 as described
in Example 5. 10.sup.5 CD3-positive T cells and 0.5 ml Eco-EGFP
virus supernatant (10.sup.5 cfu/ml) were added to each well of the
microplate for viral infection for 3 hours. RPMI 1640 medium
containing 10% FCS, 500 units/ml of recombinant mouse
interleukin-1.alpha. (Genzyme), 10 ng/ml of recombinant mouse
interleukin-2 (Genzyme), 50 units/ml of penicillin and 50 .mu.g/ml
of streptomycin was added thereto. The incubation was continued for
48 hours. After incubation, the cells were collected by using a
cell detachment buffer and washed. The gene transfer efficiency was
calculated as described in Example 4. The results are shown in
Table 9. TABLE-US-00009 TABLE 9 Functional substance Transfer
efficiency (%) BSA (control) 0.83 CH-296 8.78 CH-296/mouse laminin
13.20
Gene transfer efficiency in % is indicated.
[0116] As shown in Table 9, it was demonstrated that the efficiency
of gene transfer into mouse CD3-positive T cells was increased by
the presence of laminin.
Example 7
Involvement of Sugar Chain of Laminin Molecule in Gene Transfer
[0117] A 96-well microplate was coated using 50 .mu.l/well of a
mixture containing 5 .mu.g/ml of mouse laminin and 80 .mu.g/ml of
CH-271 as described in Example 5. The effect of treatment of the
plate with various enzymes having activities of cleaving sugar
chains on gene transfer efficiency was examined.
[0118] Plates were treated with enzymes as follows: Enzyme
solutions containing 500 mU/ml O-glycanase
(endo-.alpha.-N-acetylgalactosaminidase, Seikagaku Corp.), 500
mU/ml endoglycosidase H (endo-.beta.-N-acetylglucosaminidase H,
Seikagaku Corp.), 250 mU/ml endo-.beta.-galactosidase (Seikagaku
Corp.) and 2 mU/ml .alpha.-mannosidase (Seikagaku Corp.) in 50 mM
citrate-phosphate buffer (pH 5.0) were prepared. An enzyme solution
containing 250 mU/ml glycopeptidase F (peptide: N-glycosidase F,
Takara Shuzo) in 100 mM tris-hydrochloride buffer (pH 8.6) was
prepared. 50 .mu.l each of the enzyme solutions was dispensed in
each well for reacting at 37.degree. C. for 20 hours. The plate was
then washed three times with PBS and then used for viral infection
experiments.
[0119] 10.sup.4 mouse leukemia L1210 cells grown in RPMI 1640
medium supplemented with 10% FCS, 50 units/ml penicillin and 50
.mu.l/ml streptomycin, and 50 .mu.l of PM5neo virus supernatant
(10.sup.4 cfu/ml) were added to each well of the microplate. The
plate was incubated for 24 hours. The medium was changed to the
same medium containing G418 (Gibco) at a final concentration of
0.75 mg/ml. The plate was incubated for additional 48 hours.
G418-resistance cells were assessed as described in Example 3. The
results are shown in Table 10. Table 10 summarizes results from
three rounds of experiments. TABLE-US-00010 TABLE 10 Functional
substance Enzyme treatment Absorbance BSA (2%, control) No 0.000
.+-. 0.030 CH-271 (80 .mu.g/ml) No 1.376 .+-. 0.012 CH-271/laminin
No 1.781 .+-. 0.062 (80 .mu.g/ml:5 .mu.g/ml) CH-271/laminin
O-Glycanase 1.886 .+-. 0.071 (80 .mu.g/ml:5 .mu.g/ml)
CH-271/laminin Endoglycosidase H 1.214 .+-. 0.017 (80 .mu.g/ml:5
.mu.g/ml) CH-271/laminin E-.beta.-galactosidase 1.939 .+-. 0.083
(80 .mu.g/ml:5 .mu.g/ml) CH-271/laminin .alpha.-Mannosidase 1.657
.+-. 0.033 (80 .mu.g/ml:5 .mu.g/ml) CH-271/laminin Glycopeptidase F
1.610 .+-. 0.036 (80 .mu.g/ml:5 .mu.g/ml)
[0120] As shown in Table 10, when CH-271 was used in combination
with laminin, the appearance of G418-resistant cells was increased
as compared with the case in which CH-271 was used alone. Treatment
of the plate coated with laminin with endoglycosidase H completely
abolished the gene transfer-promoting effect of laminin.
Furthermore, treatment of the plate with .alpha.-mannosidase or
glycopeptidase F decreased the gene transfer efficiency in some
degree. According to a report concerning the sugar chains of a
laminin molecule [Biochim. Biophysi. Acta, 883:112-126 (1986)],
most of the sugar chains of the laminin molecule are N-linked sugar
chains which are bound to asparagine. 43 molecules of N-linked
sugar chains are bound to a laminin molecule. Among the sugar
chains, high mannose type asparagine-N-linked sugar chains are
released by treatment with endoglycosidase H. The fact that a
decrease in gene transfer efficiency was also observed when treated
with .alpha.-mannosidase suggests that sugar chains of the laminin
molecule play an important role. Such sugar chains have a structure
containing .alpha.1-2- and/or .alpha.1-6-bonded mannose, which is
cleaved with .alpha.-mannosidase, represented by
(Mannose).sub.9-(GlucNAc).sub.2-Asn and/or
(Mannose).sub.6-(GlucNAc).sub.2-Asn. As described above, it was
demonstrated that the gene transfer-promoting effect of laminin was
due to sugar chains of the laminin molecule, in particular high
mannose type sugar chains.
[0121] The involvement of (Mannose).sub.9-(GlucNAc).sub.2-Asn in
gene transfer was confirmed by the following experiments.
[0122] 1 g of soybean agglutinin prepared from de-fatted soybean
flour (Sigma) using Sepharose CL-2B (Pharmacia) to which lactose
had been immobilized was heat-denatured, and then digested with 20
mg of Actinase E (Kaken Pharmaceutical) in 20 ml of 50 mM
tris-hydrochloride buffer (pH 7.2) containing 10 mM calcium
chloride at 37.degree. C. for 2 days. After heat-inactivating the
enzyme, the mixture was subjected to a chromatography using
Sephadex G-15 (50 ml) column and Sephadex G-25 (150 ml) column to
purify (Mannose).sub.9-(GlucNAc).sub.2-Asn. FIG. 1 illustrates the
structure of (Mannose).sub.9-(GlucNAc).sub.2-Asn from which the
asparagine residue is removed.
[0123] A microplate to which CH-271 and
(Mannose).sub.9-(GlucNAc).sub.2-Asn were immobilized through
covalent bonds was prepared. Briefly, a 96-well Carboplate (ELISA
Carbo-type plate) (Sumitomo Bakelite) was activated using 4 mg/ml
water-soluble carbodiimide solution at 37.degree. C. for 2 hours,
and then washed three times with sterile water. 50 .mu.l each of
solutions containing 2% BSA or 80 .mu.g/ml of CH-271 as well as
(Mannose).sub.9-(GlucNAc).sub.2-Asn at a varying concentration was
added to each well of the activated 96-well Carboplate. The plate
was subjected to immobilization reaction at 37.degree. C. for 2
hours. The plate was blocked using 0.2% glycine solution at
4.degree. C. for 15 hours and then used for the following gene
transfer experiments.
[0124] 10.sup.3 L1210 cells and 0.1 ml Eco-EGFP virus supernatant
(10.sup.6 cfu/ml) were added to the well of the microplate. After
the plate was incubated for 48 hours, 0.1 ml of fresh RPMI 1640
medium containing FCS, penicillin and streptomycin was added to the
well. The plate was incubated for an additional 24 hours. The cells
were collected and washed. The gene transfer efficiency was
calculated as described in Example 4. The mean results from two
independent experiments are shown in Table 11. TABLE-US-00011 TABLE
11 Functional Concentration of sugar chain added substance No (80
.mu.g/ml) addition 2.8 .mu.g/ml 5.5 .mu.g/ml 11.1 .mu.g/ml 22.1
.mu.g/ml 44.2 .mu.g/ml 88.5 .mu.g/ml BSA (2%) 1.68 Not Not Not 1.24
1.65 Not done done done done CH-271 26.9 27.1 29.9 34.7 39.2 52.0
58.7 Gene transfer efficiency in % is indicated.
[0125] As shown in Table 11, the gene transfer efficiency for the
wells on which (Mannose).sub.9-(GlucNAc).sub.2-Asn and CH-271 had
been immobilized was increased depending on the concentration of
sugar chain used. Thus, it was confirmed that the sugar chain
having the same structure as that of the laminin molecule
contributed to the increase in gene transfer efficiency.
Example 8
Gene Transfer Specific for CD4-Positive Cells Using Anti-CD4
Monoclonal Antibody
[0126] A 24-well non-treated microplate for cell culture was coated
with a combination of 1 .mu.g/ml of an anti-mouse CD4 monoclonal
antibody or an anti-mouse CD44 monoclonal antibody (both from
Pharmingen) and 80 .mu.g/ml of H-271, CH-271 or CH-296 as described
in Example 4. H-271 was coated according to the pre-coating method
whereas CH-271 and CH-296 were coated according to the cocktail
method.
[0127] 0.5 ml of Eco-EGFP virus supernatant (10.sup.7 cfu/ml) was
added to each well of the microplate. The plate was incubated at
32.degree. C. for 3 hours, and then washed with RPMI 1640 medium
containing 10% FCS, 50 units/ml of penicillin and 50 .mu.g/ml of
streptomycin. 10.sup.5 CD3-positive T cells derived from mouse
spleen cells, prepared and pre-stimulated as described in Example 6
were added to the well for viral infection for 3 hours. Thereafter,
a RPMI 1640 medium containing 10% FCS, 500 units of recombinant
mouse interleukin-1.alpha., 10 ng/ml of recombinant mouse
interleukin-2, 50 units/ml of penicillin and 50 .mu.g/ml of
streptomycin was added to the well. The plate was incubated for an
additional 48 hours. After incubation, the cells were collected by
using a cell detachment buffer, washed and then stained with an
anti-mouse CD4 monoclonal antibody (Pharmingen) labeled with
phycoerythrin (PE; Pharmingen) and propinium iodide (PI, Sigma).
These cells were subjected to flow cytometry using FACSVantage.TM.
at an excitation wavelength of 488 nm and a emission wavelength of
515-545 nm or 562-588 nm to two-dimensionally analyze CD4 antigen
expression and EGFP expression in viable cells. The efficiencies of
gene transfer in CD4-positive cells and CD4-negative cells were
calculated. The results are shown in Table 12. Table 12 summarizes
the results from four rounds of experiments. TABLE-US-00012 TABLE
12 Efficiency of Efficiency of transfer into transfer into
Functional CD4-positive cells CD4-negative cells substance (%) (%)
BSA (control) 0.16 .+-. 0.07 0.11 .+-. 0.07 Anti-CD4 antibody 0.24
.+-. 0.19 0.12 .+-. 0.04 Anti-CD44 antibody 1.92 .+-. 0.82 1.95
.+-. 1.00 H-271 31.02 .+-. 7.34 16.54 .+-. 4.30 Anti-CD4 antibody/
58.91 .+-. 8.11 20.32 .+-. 4.46 H-271 Anti-CD44 antibody/ 56.08
.+-. 7.53 40.96 .+-. 7.04 H-271 CH-271 44.63 .+-. 6.40 26.21 .+-.
5.73 Anti-CD4 antibody/ 64.81 .+-. 9.74 25.97 .+-. 1.25 CH-271
Anti-CD44 antibody/ 60.29 .+-. 8.71 44.10 .+-. 3.56 CH-271 CH-296
48.81 .+-. 8.77 29.45 .+-. 4.70 Anti-CD4 antibody/ 62.93 .+-. 6.45
30.84 .+-. 3.27 CH-296 Anti-CD44 antibody/ 56.79 .+-. 9.87 41.37
.+-. 1.14 CH-296 (Mean .+-. standard deviation)
[0128] As shown in Table 12, when a retroviral infection was
carried out in a plate coated with both of a monoclonal antibody
and a fibronectin fragment, an effect of enhancing gene transfer
efficiency for CD3-positive T cells derived from mouse spleen cells
was observed.
[0129] Among others, it should be noted that the efficiency of gene
transfer into CD4-positive cells was much higher than that into
CD4-negative cells when viral infection was carried out using a
combination of an anti-CD4 monoclonal antibody and a functional
substance having an activity of binding to a retrovirus. For
example, the efficiency of gene transfer into CD4-positive cells
using a combination of anti-CD4 monoclonal antibody and H-271 was
very high (about 60%), while the efficiency of gene transfer into
CD4-negative cells was only about 20%. Similar results were
observed when CH-271 or CH-296 was used as a fibronectin
fragment.
[0130] On the other hand, CD44 antigen is expressed in 98% of more
of both CD4-positive cells and CD4-negative cells. Therefore, it
was expected that the gene transfer efficiency would be increased
regardless of the expression of CD4 antigen in the cells when an
anti-CD44 monoclonal antibody was used for the retroviral infection
as described above. The results in Table 12 confirm such
expectation.
Example 9
Gene Transfer Specific for CD8-Positive Cells Using Anti-CD8a
Monoclonal Antibody
[0131] Experiments were carried out as described in Example 8
except that H-271 as a functional substance having an activity of
binding to a retrovirus, and an anti-mouse CD8a monoclonal antibody
(Pharmingen) and an anti-mouse CD44 monoclonal antibody as
antibodies were used. An anti-mouse CD8a monoclonal antibody
(Pharmingen) labeled with phycoerythrin (PE; Pharmingen) was used
for detecting CD8-positive and CD8-negative cells. The results are
shown in Table 13. Table 13 summarizes the results from two rounds
of experiments. TABLE-US-00013 TABLE 13 Efficiency of Efficiency of
transfer into transfer into Functional CD8-positive cells
CD8-negative cells substance (%) (%) BSA (control) 0.22 .+-. 0.08
0.28 .+-. 0.00 Anti-CD8a antibody 0.36 .+-. 0.20 0.28 .+-. 0.02
Anti-CD44 antibody 0.98 .+-. 0.34 0.92 .+-. 0.20 H-271 20.08 .+-.
4.71 26.43 .+-. 6.07 Anti-CD8a antibody/ 36.07 .+-. 1.57 24.42 .+-.
0.55 H-271 Anti-CD44 antibody/ 46.93 .+-. 0.88 47.16 .+-. 0.75
H-271 (Mean .+-. standard deviation)
[0132] As shown in Table 13, an effect of enhancing the efficiency
of gene transfer into CD3-positive T cells derived from mouse
spleen cells was observed when the combination of an anti-CD8a
monoclonal antibody and a fibronectin fragment was used.
[0133] When the anti-CD8a monoclonal antibody was used, high gene
transfer efficiency for cells expressing CD8 antigen recognized by
the antibody was observed (Example 8). On the other hand, when a
monoclonal antibody against CD44 which is expressed in 98% of more
of both CD8-positive cells and CD8-negative cells was used, no
difference in gene transfer efficiency was recognized between
CD8-positive cells and CD8-negative cells.
[0134] The experimental results in Examples 8 and 9 are very
significant. These results demonstrate that a gene of interest can
be transferred specifically into target cells if a cell population
containing target cells is infected with a retrovirus containing
the gene of interest in a culture vessel which has been coated
using a mixture (cocktail) of an antibody which specifically binds
to the target cells and a functional substance having an activity
of binding to the virus.
Example 10
Cell-Specific Gene Transfer Using an Antibody
[0135] A 24-well non-treated microplate for cell culture was coated
as described in Example 4 according to the cocktail method using 80
.mu.g/ml of CH-271 and 1 .mu.g/ml of one of the monoclonal
antibodies against various cell surface antigens (anti-CD4,
anti-CD8, anti-CD44, anti-CD49c, anti-CD49d and anti-CD49e
antibodies; all from Pharmingen).
[0136] K562 (human chronic myelogenous leukemia cell, ATCC
CCL-243), HSB-2 (human acute lymphoblastic leukemia cell,
CCRF-HSB-2, ATCC CCL-120.1), MOLT-3 (human acute lymphoblastic
leukemia cell, ATCC CRL-1552) and TF-1 (human erythroleukemia cell,
ATCC CRL-2003) were used as target cells. FACS analysis was carried
out on these cells using labeled monoclonal antibodies to determine
the expression of antigens corresponding to the antibodies.
[0137] 0.5 ml of Ampho-EGFP virus supernatant (1.times.10.sup.6
cfu/ml) was added to each well of the microplate. The plate was
incubated at 32.degree. C. for 3 hours, and then washed with RPMI
1640 medium containing 10% FCS, 50 units/ml of penicillin and 50
.mu.g/ml of streptomycin. 1.times.10.sup.5 of each of the
respective cells suspended in 1 ml of the medium was added to the
well for viral infection. After incubating for additional 3 days,
the cells were collected by using a cell detachment buffer and
washed. The efficiency of EGFP gene transfer was calculated
according to the flow cytometry method as described in Example
4.
[0138] The results are shown Table 14. The mean results from three
independent experiments are shown. TABLE-US-00014 TABLE 14 Cells
used HSB-2 MOLT-3 TF-1 K562 Transfer CD ag Transfer CD ag Transfer
CD ag Transfer CD ag Antibody eff. exp. eff. exp. eff. exp. eff.
exp. used (%) ratio (%) ratio (%) ratio (%) rate None 100 100 100
100 CD4 106.7 - 100.7 +/- 108.8 +/- 104.9 - CD8 130.4 ++ 130.4 ++
107.0 - 116.9 - CD44 173.7 ++ 172.5 ++ 188.9 +++ 135.1 - CD49c
153.9 +++ 102.7 - 115.6 - 106.3 - CD49d 159.2 ++ 165.3 +++ 150.3
+++ 97.5 - CD49e 185.5 +++ 127.5 + 128.9 ++ 172.6 +++ Gene transfer
efficiency (Transfer eff.) is expressed as relative value (%)
assuming the efficiency of gene transfer without the addition of an
antibody for the respective cells as 100%. CD antigen expression
ratios (CD ag exp. ratio) represent the ratios of positive cells
(%) in FACS measurements as follows: -: 10% or less; +/-: 10-30%;
+: 30-60%; ++: 60-90%; +++: 90% or more.
[0139] As shown in Table 14, the antigen expression ratio
correlated with the efficiency of gene transfer using the cocktail
method in which CH-271 as a virus-binding substance and the
antibody against the antigen on the cell as a cell-binding
substance were used.
[0140] Furthermore, gene transfer experiments were carried out
using 80 .mu.g/ml of polylysine as a functional substance having an
activity of binding to a retrovirus in place of CH-271. Monoclonal
antibodies and cells used, as well as other experimental
conditions, were as described above. The results are shown in Table
15. The mean results from three independent experiments are shown.
TABLE-US-00015 TABLE 15 Cells used HSB-2 MOLT-3 TF-1 K562 Transfer
CD ag Transfer CD ag Transfer CD ag Transfer CD ag Antibody eff.
exp. eff. exp. eff. exp. eff. exp. used (%) ratio (%) ratio (%)
ratio (%) rate None 100 100 100 100 CD4 103.3 - 104.1 +/- 98.6 +/-
99.4 - CD8 116.3 ++ 136.7 ++ 100.8 - 92.4 - CD44 155.5 ++ 144.9 ++
253.1 +++ 102.6 - CD49c 160.1 +++ 104.7 - 116.1 - 100.6 - CD49d
138.2 ++ 156.3 +++ 187.7 +++ 103.1 - CD49e 142.5 +++ 140.0 + 166.1
++ 129.2 +++ Gene transfer efficiency (Transfer eff.) is expressed
as relative value (%) assuming the efficiency of gene transfer
without the addition of an antibody for the respective cells as
100%. CD antigen expression ratios (CD ag exp. ratio) represent the
ratios of positive cells (%) in FACS measurements as follows: -:
10% or less; +/-: 10-30%; +: 30-60%; ++: 60-90%; +++: 90% or
more.
[0141] As shown in Table 15, the antigen expression ratio
correlated with the efficiency of gene transfer using the cocktail
method in which polylysine as a virus-binding substance and the
antibody against the antigen on the cell as a cell-binding
substance were used.
[0142] Both series of experimental results as described above
demonstrate that a gene can be transferred specifically into target
cells of interest by transferring a gene according to the cocktail
method and using an antibody that specifically recognizes an
antigen expressed on the target cell as a cell-binding
substance.
Example 11
Gene Transfer into Target Cells Pre-Cultured in Medium Containing
Deferoxamine
[0143] Human myelocytic leukemia HL-60 cells (ATCC CCL-240)
cultured in RPMI 1640 medium containing 10% FCS, 50 units/ml of
penicillin and 50 .mu.g/ml of streptomycin were transferred into
the same medium containing deferoxamine (Sigma) at a varying
concentration on the day before the infection experiments. The
cells were cultured at 37.degree. C. for 20 hours in the presence
of 5% CO.sub.2. The cells were washed with fresh medium without
deferoxamine, and then suspended at a concentration of
2.times.10.sup.5 cells/ml for use in the following infection
experiments.
[0144] 0.5 ml of 80 .mu.g/ml CH-271 was added to each well of a
24-well non-treated microplate for cell culture. The plate was
allowed to stand at 4.degree. C. overnight, blocked using 2% BSA
for 30 minutes and washed with PBS. 0.5 ml of Ampho-EGFP virus
supernatant (10.sup.6 cfu/ml) was added to the well of the
microplate. The plate was incubated at 32.degree. C. for 3 hours
and washed with RPMI 1640 medium containing 10% FCS, 50 units/ml of
penicillin and 50 .mu.g/ml of streptomycin. 10.sup.5 of the
pre-cultured HL-60 cells were added to the well. The plate was
incubated for 48 hours. 0.5 ml of RPMI 1640 medium containing 10%
FCS, 50 units/ml of penicillin and 50 .mu.g/ml of streptomycin was
added to the well. The plate was incubated for an additional 24
hours. Thereafter, the gene transfer efficiency was determined as
described in Example 4. The results are shown in Tables 16 and 17.
TABLE-US-00016 TABLE 16 Deferoxamine Transfer concentration
Functional efficiency (.mu.g) substance (%) No addition BSA
(control) 0.01 No addition CH-271 0.14 6.25 CH-271 0.22 12.5 CH-271
0.27 25 CH-271 0.35 50 CH-271 0.71
[0145] TABLE-US-00017 TABLE 17 Deferoxamine Transfer concentration
Functional efficiency (.mu.g) substance (%) No addition BSA
(control) 0.02 No addition CH-271 0.25 40 CH-271 11.14
[0146] As shown in Tables 16 and 17, an increase in gene transfer
efficiency was observed even for HL-60 cells by pre-treating the
cells with deferoxamine for 20 hours. It was known that a gene is
transferred into HL-60 cells with very low efficiency using CH-271
alone.
Example 12
Detection of Presence of Viral Infection-Inhibitory Substances in
Culture Supernatant
[0147] The TKNeo virus supernatant prepared in Example 2 was
diluted with DMEM, a culture supernatant of NIH/3T3 cells (ATCC
CRL-1658) or a culture supernatant of .phi.CRIP cells to a
concentration of 312.5 cfu/ml for use in the following
procedures.
[0148] 0.5 ml of 32 .mu.g/ml CH-296 was added to each well of a
24-well non-treated microplate for cell culture. The plate was
allowed to stand at room temperature for 2 hours, blocked with 2%
BSA for 30 minutes and washed with PBS. 1 ml of the above-mentioned
virus supernatant and 2.times.10.sup.4 NIH/3T3 cells were added to
the well of the plate. The plate was incubated at 37.degree. C.
overnight. The cells were then cultured in a selective medium
containing 0.75 mg/ml of G418 for 10 days. The number of colonies
formed was counted. The ratio of the number of G418-resistant
colonies to the number of colonies formed in a medium without G418
was defined as gene transfer efficiency. The results are shown in
Table 18. TABLE-US-00018 TABLE 18 Gene transfer efficiency Diluent
(%) DMEM (control) 100 NIH/3T3 cell 20.6 culture supernatant
.phi.CRIP cell 15.7 culture supernatant
[0149] As shown in Table 18, the gene transfer efficiencies were
decreased to one fifth or less when the dilution of virus with the
culture supernatant of NIH/3T3 cells or the culture supernatant of
.phi.CRIP cells was used as compared with the efficiency of gene
transfer by dilution with DMEM. NIH/3T3 cell is the parent strain
of many packaging cell lines such as .phi.CRIP cell and GP+EmvAm12
cell, which was used to generate the producer cell for the TKNeo
virus vector used in this experiment. The fact that an activity of
inhibiting retroviral infection was found in the culture
supernatants of these cells suggests that virus supernatants
prepared using similar packaging cells also contain inhibitory
substances.
Example 13
Removal of Viral Infection-Inhibitory Substance in Virus
Supernatant
[0150] The following procedures were used to remove the viral
infection-inhibitory substance found in Example 12. The TKNeo virus
supernatant prepared in Example 2 was diluted with the culture
supernatant of .phi.CRIP cells to a concentration of 5000 cfu/ml.
The diluted supernatant was further doubly diluted with DMEM for
use as a sample containing a retrovirus.
[0151] 1 ml of the virus supernatant was added to each well of a
plate coated with CH-296 as described in Example 11. The plate was
incubated for 1 to 5 hours for contacting and binding the virus
particles with CH-296. The plate was then washed three times with
PBS. 1 ml of DMEM containing 2.times.10.sup.4 NIH/3T3 cells was
added to the well. As a control, 2.times.10.sup.4 NIH/3T3 cells
were suspended in 1 ml of the above-mentioned virus supernatant and
then immediately transferred to the plate coated with CH-296. These
plates were incubated at 37.degree. C. overnight to allow the virus
to infect cells. After infection, the cells were cultured in a
selective medium containing 0.75 mg/ml of G418 for 10 days, and the
number of colonies formed were counted. The ratio of the number of
G418-resistant colonies to the number of colonies formed in a
medium without G418 was defined as gene transfer efficiency. The
results are shown in FIG. 3.
[0152] As shown in FIG. 3, higher transfer efficiency was observed
at 3 hours when virus particles were contacted with and bound to
the CH-296 coated plate as compared with the transfer efficiency
for the control group. Thus, it was demonstrated that the activity
of inhibiting viral infection in a virus supernatant could be
removed by the procedures described above.
Example 14
Removal of Sodium Butyrate in Virus Supernatant
[0153] Recombinant retrovirus-producer cells obtained by
introducing a retrovirus vector plasmid PLEIN into .phi.CRIP cells
were cultured in DMEM containing 10% CS. When the cells grew to
semi-confluence in a 10-cm plate, the medium was changed to 7 ml of
RPMI 1640 containing 10% FCS or 7 ml of RPMI 1640 containing 5 mM
sodium butyrate (Nacalai Tesque) and 10% FCS. After the cells were
cultured for 24 hours, the supernatants were filtered through 0.45
.mu.m filters to obtain virus supernatants. The titer of the virus
supernatant was determined as described in Example 2. The titer of
the virus supernatant without sodium butyrate was
3.3.times.10.sup.4 cfu/ml, whereas the titer of the virus
supernatant containing 5 mM sodium butyrate was 2.times.10.sup.6
cfu/ml.
[0154] Sodium butyrate has the activities of arresting cell cycle
to suppress cell growth and of inducing differentiation. Thus, it
may have a harmful influence on infected cells. Removal of sodium
butyrate contained in a virus supernatant was assessed as
follows.
[0155] HL-60 cells were used as target cells. 0.5 ml of the
above-mentioned virus supernatant was added to each well of a plate
coated with CH-296 as described in Example 12. The plate was
incubated at 37.degree. C. for 3 hours for contacting and binding
the virus particles with CH-296. After incubation, the plate was
washed three times with PBS. 0.5 ml of RPMI 1640 medium
supplemented with 10% FCS containing 5.times.10.sup.4 HL-60 cells
was added to the well. As a control, 5.times.10.sup.4 HL-60 cells
were suspended in 0.5 ml of the above-mentioned virus supernatant
and then immediately added to the plate coated with CH-296. These
plates were incubated at 37.degree. C. overnight to allow the virus
to infect cells. After the incubation, 1 ml of RPMI 1640 medium
containing 10% FCS was added to the well. The plate was incubated
for additional 48 hours. The cell number was then counted.
Furthermore, EGFP-expressing cells were detected according to the
flow cytometry method as described in Example 4 to analyze the gene
transfer efficiency. The results are shown in Table 19.
TABLE-US-00019 TABLE 19 Experimental group/ Cell number Gene
transfer virus supernatant (cells/plate) efficiency (%) CH-296
Sodium butyrate: - 2.0 .times. 10.sup.5 2.21 Sodium butyrate: + 1.8
.times. 10.sup.5 52.98 Control Sodium butyrate: - 1.7 .times.
10.sup.5 2.74 Sodium butyrate: + 4.0 .times. 10.sup.5 35.46
[0156] As shown in Table 19, higher gene transfer efficiency was
observed for the control group when a virus supernatant prepared by
adding sodium butyrate was used, confirming the effectiveness of
sodium butyrate in the virus preparation. However, the viable cell
number observed using the supernatant containing sodium butyrate
was one fourth or less of that observed using the supernatant
without sodium butyrate, confirming that sodium butyrate suppressed
cell growth. On the other hand, when the virus particles were
contacted with and bound to the CH-296 coated plate beforehand, the
cell growth suppression, although observed for control group using
the supernatant containing sodium butyrate, was not observed. An
increase, rather than a decrease, in gene transfer efficiency was
observed. Thus, it was demonstrated that high gene transfer
efficiency could be achieved without the influence of sodium
butyrate by contacting virus with CH-296 followed by washing.
[0157] Next, DEAE-dextran was used to carry out similar
experiments.
[0158] DEAE-dextran (Sigma) was dissolved at a concentration of 10
mg/ml in PBS. The solution was sterilized by filtering through a
0.22 .mu.m filter and used to coat a plate. 1.1 ml of a mixture
obtained by mixing 10 volumes of PBS and 1 volume of the
DEAE-dextran solution was added to each well of a 6-well
non-treated plate for cell culture (Iwaki Glass). The plate was
incubated at 4.degree. C. overnight. The DEAE-dextran solution was
removed from the plate. 2 ml of 2% BSA solution was added to the
well for treatment for 30 minutes. The plate was washed three times
with 2 ml/well of PBS. As a control, a plate was prepared by
conducting the same procedures except that PBS was used in place of
the DEAE-dextran solution.
[0159] A virus supernatant was prepared according to the method in
which sodium butyrate was added as described above using
recombinant retrovirus-producer cells produced by introducing a
retrovirus vector plasmid PLEIN into GP+E86 cells [J. Virol.,
62:1120-1124 (1988)]. 1 ml of a virus dilution (1.6.times.10.sup.6
cfu/ml) obtained by diluting 1 volume of the virus supernatant with
20 volumes of DMEM containing 10% CS was added to each well. The
plate was incubated at 37.degree. C. for 2 hours, and then washed
three times with 2 ml/well of PBS. 5.times.10.sup.4 NIH/3T3 cells
were added to the well. The plate was incubated at 37.degree. C.
for 3 days in the presence of 5% CO.sub.2. After incubation, the
cells were treated with trypsin and collected. EGFP-expressing
cells were analyzed according to the flow cytometry method as
described in Example 4 to determine the gene transfer efficiency.
The results are shown in Table 20. TABLE-US-00020 TABLE 20 Coating
Gene transfer efficiency (%) DEAE-dextran 26.7 Control 0.7
Gene transfer efficiency represented by the ratio (%) of
EGFP-positive cells to total cells is indicated.
[0160] As shown in Table 20, it was demonstrated that DEAE-dextran
also has an activity of binding to a retrovirus, and it can be used
for the gene transfer method of the present invention.
Example 15
Binding of Functional Substance to Retrovirus Utilizing
Centrifugation Method
[0161] .phi.CRIP cells into which a retrovirus plasmid, DOL vector,
containing a neomycin-resistance gene [Proc. Natl. Acad. Sci. USA,
84:2150-2154 (1987)] had been introduced were cultured in DMEM
containing 10% CS, 50 units/ml of penicillin and 50 .mu.g/ml of
streptomycin. A DOL virus supernatant was prepared as follows.
Medium in a 10-cm plate in which the producer cells had grown to
semi-confluence was changed to 5 ml of DMEM containing 10% CS.
After 24 hours, the supernatant was collected and filtered through
a 0.45 .mu.m filter (Millipore). The titer of the virus supernatant
was 8.7.times.10.sup.5 cfu/ml.
[0162] A centrifugation tube (50 ml polypropylene conical tube,
Falcon) used for infecting cells with a retrovirus was coated with
CH-296 as follows. Briefly, 3 ml of PBS containing 40 .mu.g/ml of
CH-296 was slowly placed to the bottom of the centrifugation tube.
The tube was allowed to stand upright for incubation at 4.degree.
C. for 16 hours. The CH-296 solution was exchanged for 3.5 ml of
PBS containing 2% BSA. The tube was incubated for additional 30
minutes at room temperature, and then washed with 5 ml of Hanks'
balanced salt solution (HBSS, Gibco).
[0163] The DOL retrovirus was bound to the bottom of the
centrifugation tube coated with CH-296 as follows. Briefly, 5 ml of
the undiluted DOL virus supernatant, or a 10-fold or 100-fold
dilution thereof was placed in the centrifugation tube. The tube
was centrifuged at 2900.times.g at 25.degree. C. for 3 hours to
force the retrovirus to bind to CH-296. For comparison, the
above-mentioned virus supernatant was added to a 6-well non-treated
plate for cell culture (Falcon) coated with CH-296 using PBS
containing 40 .mu.g/ml of CH-296 so as to result in a density of 8
.mu.g/cm.sup.2. The plate was allowed to stand at 37.degree. C. for
4 hours for binding and used in the following procedures.
[0164] Gene transfer into NIH/3T3 cells was carried out using the
centrifugation tube coated with CH-296 to which the retrovirus had
been forced to bind by centrifugation. Briefly, 1.times.10.sup.5
NIH/3T3 cells were placed in the centrifugation tube coated with
CH-296 in which one of the serial dilutions of the virus
supernatant had been centrifuged. The tube was incubated at
37.degree. C. for 3 hours (hereinafter referred to as the
centrifugation method). Alternatively, the above-mentioned
microplate was incubated under the same conditions (hereinafter
referred to as the binding method). As a control, a mixture of the
virus supernatant and NIH/3T3 cells was added to the microplate
coated with CH-296, and the plate was incubated at 37.degree. C.
for 3 hours. The results obtained using the last method were
defined as those of a conventional infection method and used for
comparison (hereinafter referred to as the supernatant method).
After incubation, the cells were collected. The efficiency of gene
transfer in the collected cells was determined as described in
Example 13. The results are shown in FIG. 4. In FIG. 4, the
horizontal axis represents the dilution rate of the virus
supernatant and the vertical axis represents the gene transfer
efficiency. The open bars, shaded bars and closed bars represent
results obtained using the supernatant method, the binding method
and the centrifugation method, respectively.
[0165] As shown in FIG. 4, the efficiency of gene transfer using
the centrifugation method was higher than that using the
conventional supernatant method or the method in which the virus
had been spontaneously adsorbed to CH-296 prior to the infection
(the binding method). Thus, it was demonstrated that more virus
particles bound to CH-296 at the bottom of the vessel by forcing
the virus to precipitate by centrifugal force. In particular, the
effect due to the centrifugation was remarkable when a diluted
virus supernatant was used.
[0166] Furthermore, the titers of the virus supernatants collected
after the binding of virus using centrifugation or standing
(binding) were measured. The results are shown in Table 21.
TABLE-US-00021 TABLE 21 Virus titer Recovery after Sample (cfu/ml)
binding (%) Virus supernatant 8.7 .times. 10.sup.5 100 (before use)
Collected supernatant - 7.8 .times. 10.sup.5 89.4 binding method
Collected supernatant - 7.6 .times. 10.sup.4 8.8 centrifugation
method
[0167] As shown in Table 21, in the case of standing, the collected
supernatant had about 80 to 90% of the titer of the supernatant
before binding. On the other hand, the titer of the supernatant
after forcing to bind by centrifugation was one tenth or less of
that of the supernatant before binding. These results demonstrate
that more virus particles were bound to CH-296 by centrifugal
force. PBS used to wash the centrifugation tube after
centrifugation contained about 2% of the original amount of the
virus. In addition, almost the same gene transfer efficiency was
observed regardless of the presence of the washing step. These
results suggest that the virus particles were firmly held by CH-296
when centrifugation was utilized.
[0168] Furthermore, the efficiency of gene transfer by the
centrifugation method was compared with that by a method in which
cells are infected with a virus during centrifugation.
[0169] The efficiency of gene transfer into NIH/3T3 cells by the
centrifugation method was compared with that by the
centrifugation-infection method, in which a virus is precipitated
by centrifugal force onto cells for infection (see WO 95/10619). 5
ml of a virus supernatant prepared by diluting the virus
supernatant prepared using GP+E86 cells as described in Example 14
with a culture supernatant of NIH/3T3 cells to a concentration of
1.times.10.sup.5 cfu/ml was used for the comparison. Briefly, gene
transfer was carried out as follows. The above-mentioned virus
supernatant was added to a centrifugation tube coated with CH-296.
The tube was centrifuged at 30.degree. C. at 2900.times.g for 4
hours, and then washed with PBS. Cells were then added to the tube
for infection at 37.degree. C. for 4 hours (the centrifugation
method). Cell were added to a centrifugation tube coated with
CH-296, and cultured for 2 hours. The virus supernatant was added
to the tube. The tube was centrifuged at 30.degree. C. at
2900.times.g for 4 hours for infection (the
centrifugation-infection method). In these methods, the
centrifugation tubes were coated with CH-296 as described above,
and 1.times.10.sup.5 NIH/3T3 cells were used for gene transfer. The
cells after infection were re-plated into a 60-mm plate, and
cultured for 2 days. The efficiency of the EGFP gene transfer was
then determined according to the flow cytometry method as described
in Example 4. The results are shown in FIG. 5.
[0170] As shown in FIG. 5, it was demonstrated that the efficiency
of gene transfer by the centrifugation method was higher than that
by the centrifugation-infection method. This is considered to be
because infection-inhibitory substance in the virus supernatant was
removed by washing.
* * * * *