Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS. AND COMPOSITIONS FOR GENETICALLY
MODIFYING PRIMATE BONE MARROW CELLS
to
Field of the invention
The invention concerns the field of gene therapy and more particularly relates
to a
method for genetically modifying primate bone marrow cells so that they have
the capacity to
regenerate in vivo, and to cells that produce recombinant retroviral vectors
that can be used in
t 5 such a method. The method is exemplified by the use of means which enhance
the local
concentration of retroviral particles derived from the marine leukemia virus
in the vicinity of
target primate stem cells.
Back ound
2o Developments in the field of molecular biology have ied to a better
understanding of the
genetic basis underlying the .development of a large number of disorders. It
is expected that the
genes which are associated vrith the diseases that occur most frequently will
have been
identified, cloned and characterized before the end of this century.
So far, molecular genetics has contributed to medicine by the development of
diagnostic
25 tools and methods and the biotechnological production of pharmaceuticals.
It may be expected,
however, that it will also be possible to use the increasing knowledge of
genetics for an
essentially new therapeutic tt~eatment, the so-called gene therapy. The
purpose of gene therapy
is to treat disorders by genetically modifying somatic cells of patients. The
uses of gene therapy
are not limited to hereditary .disorders; the treatment of acquired diseases
is also considered to
3o be one of the possibilities. Although this field of study is still in a
preliminary stage and must be
developed, therapeutic possilbilities are in the distance which can
drastically improve medicine
in the future (Awderson, ( 19f~4) Science 226:410; Belmont and Caskey, ( I
986) in Gene Transfer,
R. Kucherlapati, eds, Pienurn press, New York and London, P. 411; and
Williamson, (1982)
Nature 298:416.)
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An important cell type for gene therapy purposes is the so-called
hematopoietic stem cell
which is the precursor cell of all circulating blood cells. This stem cell can
multiply itself
without losing its differentiating ability. In adult animals most stem cells
are situated in the
bone marrow. Very infrequently stem cells also start to circulate in the
peripheral blood. This
can be significantly augmented by treatment with stem cell mobilizing agents,
including but not
restricted to certain hematopoietic growth factors. In embryos, stem cells are
by nature
circulating much more frequently. Thus, bone marrow, peripheral blood after
stem cell
mobilization and embryonic blood, e.g. collected perinatally from the
umbilical vein, are useful
sources for stem cells. The underlying idea of a gene therapy directed to
hematopoietic stem
to cells is that gene transfer to (a limited number of) stem cells may already
be sufl~cient to replace
the entire blood-forming tissue with genetically modified cells for a lifetime
(Williamson,
( 1982) Nature 298:416). This would enable treatment not only of diseases that
are caused by a
(hereditary) defect of blood cells, but also of diseases that are based on the
inability to make a
certain protein: the modified blood (forming) system could be a constant
source of the protein,
which could do its work at the places where necessary. It is also possible,
with the introduction
of genetic material into the blood system, to obtain resistance against
infectious agents, to
combat cancer, or even to overcome a predisposition to chronic diseases, such
as rheumatism or
diabetes. Finally, it is noted that in the treatment of some diseases it is to
be preferred or
necessary that the gene transfer to stem cells is performed on bone marrow
cell populations from
2o which certain cell types have been removed. One could for instance consider
the use of gene
therapy in the treatment of leukemia, in which case there should not occur any
gene transfer to
the leukemic cells.
One of the conditions for providing of such a bone marrow gene therapy
protocol is a
technique by which genes can be incorporated into the chromosomes of target
cells, in such a
manner that those genes are also passed on to the daughter cells and that the
desired protein
product is produced in those cells. In the invention described here, for this
purpose use is made
of recombinant retroviruses that carry with them the genes to be introduced
and which are
capable of delivering them to mammalian cells. They make use of the natural
characteristic of
retroviruses to integrate efficiently and stably into the genome of the
infected cell, but not
3o themselves to cause a productive infection because the retroviruses used
are replication-
defective and are not contaminated with wild-type viruses (Temin, (1986) in
Gene Transfer, R.
Kucherlapati, eds. Plenum Press, New York, p. 149 and Temin, ( 1990) Hum. Gene
Ther. 1:111 ).
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The recombinant retroviruses which are used within the framework of the
present invention are
derived from viruses with a natural host-specificity that includes primates,
or from viruses that
can be pseudotyped with a host-specificity that includes primates. Said
viruses include, but are
not restricted to, marine leukemia viruses (MuLV; Weiss et al., (1984) RNA
Tumor Viruses,
New York) with a so-called amphotropic or xenotropic host-range, gibbon ape
leukemia viruses
(GaLV; Lieber et al., Proc. Natl. A~;ad. Sci. USA 72( 1975) 2315-2319), and
primate lentiviruses.
For the production of recombinant retroviruses, two elements are required: the
so-called
retroviral vector, which, ire addition to the gene (or genes) to be
introduced, contains all DNA
elements of a retrovirus that are necessary for packaging the viral genome and
the integration
1 o into the host genome; and the so-called packaging cell line which produces
the viral proteins
that are necessary for building up an infectious recombinant retrovirus
(Miller, ( 1990), Hum.
Gene Ther. 1:5).
As the presence of replication-competent viruses in a gene therapy protocol is
considered
highly undesirable, most modern packaging cell lines are constructed in a way
such that the risk
15 of recombination events whereby a~ replication-competent virus is
generated, is minimized. This
is effected by physically separating; into two parts the parts of the virus
genome that code for
viral proteins and introdu<;ing them into the cell line separately (Danos and
Mulligan, (1988)
Proc. Natl. Acad. Sci. US.4 85:6460; Markowitz et al., (1988) J. Virol.
62:1120; and Markowitz
et al., ( 1988) Virology 16'7:400).
2o As the presence of both constructs is essential to the functioning of the
packaging cell
line and chromosomal instability occurs regularly, it is of great importance
for the routine use of
such cells in gene therapy procedures that, by means of a selection medium,
selection for the
presence of the constructs. can be provided for. Therefore, these constructs
are often introduced
by means of a so-called cotransfection whereby both viral constructs are
transfected together
25 with a dominant selection marker. The possibility of selection thus
provided is certainly not a
trivial requirement, considering for instance the observation that we and
various other research
groups made, that virus-producing cells based on the packaging cell line
~rCRIP (Danos and
Mulligan, ( 1988) Proc. N'atl. Acact Sci. USA 85:6460) are not stable. That is
to say that they are
no longer resistant to the relevant selection media and during cultivation
lose their capacity to
3o produce retroviruses. One example, of importance for one of the embodiments
of the present
invention, is the so-called POC-1 cell line which was produced by us on the
basis of y~CRIP
cells (Van Beuschem et crl., (1990) J. Exp. Med 172:729) which on account of
its instability
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cannot be used for gene therapy on a routine basis. Therefore, in the
invention described here,
use is made of packaging cells which, by means of a dominant selection
culture, do continue to
produce stable virus.
Studies in mice have demonstrated that using amphotropic retroviral vectors,
bone
s marrow stem cells can be provided with a new gene. After transplantation of
these modified
cells into lethally irradiated mice, the new gene could also be demonstrated
for long periods in
many different blood cell types of the transplanted animals (Van Beuschem et
al., ( 1990) J. Exp.
Med.172:729).
Previous problems with regard to the non-expression of the newly introduced
genes were
1 o solved by us by using a retroviral vector in which the expression of the
gene of choice, is driven
by a retroviral promoter whose expression-specificity has been changed by
means of a
replacement of the so-called enhancer (Van Beuschem et al., ( 1990) J. Exp.
Med 172:729; and
Valerio et al., (1989) Gene 84:419). In the present invention, these vectors
are called LgXL
(OlVlo+pyF 101 ), wherein X represents the code of a gene yet to be filled in.
t 5 Before the results obtained from research into gene transfer into the
blood-forming organ
of mice can be translated into an eventual use of gene therapy in the clinic,
a number of essential
questions must be answered by studying a relevant preclinical model. First of
all, it has to be
demonstrated that effcient gene transfer is also possible to blood-forming
stem cells of higher
mammals, in particular primates. Moreover, genetic modification coupled with
autologous bone
2o marrow transplantation in primates requires complex logistics which cannot
be studied in mice.
The organization of the blood-forming organs of mice and humans can only be
compared to a
certain extent and it will be clear that the sizes of the two species, and
hence the numbers of
cells involved in transplantation, differ considerably.
The experimental animal model that. is eminently suitable for preclinical gene
therapy
25 studies is the nonhuman primate, in particular the rhesus monkey, partly
because the current
bone marrow transplantation protocols in the clinic are principally based on
data obtained from
experiments with bane marrow from the rhesus monkey. Gene therapy procedures
using bone
marrow cells can be tested in this animal model by taking bone marrow,
modifying this
genetically by means of recombinant retroviruses and subsequently
transplanting it back
3o autologousIy (i.e. into the same monkey) after the endogenous bone marrow
cells have been
eradicated by means of irradiation.
To date, such experiments have met with little success with regard to:
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a) the hematopoietic regeneration that could be effected with the infected
bone
marrow, and
b) the in vivo stability of the genetic modification.
In the studies published to date, gene transfer was performed either by
incubating bone
s marrow cells with cell-free virus supernatant harvested from virus-producing
cells or by a direct
exposure of the bone marrow cells to the virus-producing cells. The latter
method involves a so-
called cocultivation wherein the vinrs-producing cells are adhered to the
bottom of a culture
bottle and the bone marrow cells are; seeded on top thereof. Following
cocultivation, the non-
adherent bone marrow cell:. are subsequently harvested and used as
transplants.
1 o In the first reported study (Anderson et al., ( 1986), Gene transfer and
expression in
nonhuman primates using retroviral vectors, In Cold Spring Harbor Symposia on
Quantitative
Biology, Volume LI, eds. C'.old Sprung Harbor Laboratory, New York, p. 1073;
and Kantoff, P.
W., A. P. Gillio, J. R. Mch.achlin, C. Bordignon, et al., (1987) J. Exp. Med.
166:219), in 19
monkeys an autologous trmsplantation was performed with bone marrow cells
infected with
1 s different retroviraI vectors ~~ontaining the gene for neomycin resistance
(neo') or dihydrofolate
reductase (DHFR), or with a virus ui which neo' and the gene for adenosine
deaminase (ADA)
are located together, produced by cells that also produce replication-
competent virus. Both gene
transfer methods described above, i.e., the cocultivation procedure and the
infection with virus
supernatant that can be harvested from the virus-producing cells were
utilized. Using the
2o cocultivation procedure, it was not possible to obtain hematopoietic
regeneration after
autologous transplantation. As a result, only three out of the 13 monkeys
survived this
procedure. None of the surviving monkeys showed any signs of genetic
modification in vivo.
Complete hematopoietic reconstitution could be obtained in the six monkeys
that received
supernatant-infected bone rnarrow and in four of these animals the gene could
be demonstrated.
2s However, genetic modification remained low and transient. Nor could it be
precluded that the
observed modification had occurred in long-living T-cells which did not
generate from the bone
marrow cultured in vitro, but were already present as a contaminant in the
infected bone
marrow.
In the second study (Bodine et al., (1990) Proc. Natl. Acad Sci. USA 87:3738)
bone
3o marrow from rhesus monkeys was cocultivated with cell lines that produce
neo'-containing
viruses. In this study, also, only the provirus could be demonstrated in vivo
after infection by
means of a virus-producing cell line that produces contaminatory helper
viruses. In this setting,
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no long-term studies could be performed because again the bone marrow proved
incapable of
reconstituting the hematopoietic system.
In conclusion, in the data published so far, the cocultivation method has
always been
associated with a drastic loss of in vivo regenerating capacity of the bone
marrow cells
(Anderson et al., ( 1986), "Gene transfer and expression in nonhuman primates
using retroviral
vectors", In Cold Spring Harbor Symposia on Quantitative Biology, Volume LI,
eds. Cold
Spring Harbor Laboratory, New York, p. 1073; Kantoff, P. W., A. P. Gillio, J.
R. McLachlin, C.
Bordignon, et al., (1987) J. Exp. Med. 166:219; and Bodine et al., (1990)
Proc. Natl. Acad Sci.
USA 87:3738), so that a clinical application is precluded.
1 o In addition, none of the studies published to date are sui~ciently
interpretable as regards
genetic modification, since they invariably involved the use of virus
preparations in which
replication-competent virus was present. Via a so-called "rescue", this may
lead to a spread of
the recombinant virus genome after the cells have been transplanted, so that
it remains unclear
whether the modified cells are offspring of infected bone marrow cells. The
present invention
provides a method for efficient gene transfer into primate hematopoietic stem
cells without a
significant loss of the in vivo regenerating capacity of the isolated cells.
Relevant Literature
Fibronectin as a single molecule has been reported to bind retroviruses and
haemopoietic
2o cells, thereby enhancing the gene transfer efficiency (WO 95/26200).
SUMMARY
The invention provides a method for genetically modifying primate
hematopoietic stem
cells. The method includes the step of combining isolated primate
hematopoietic stem cells
with a recombinant retrovirus using means which increase the local
concentration of
recombinant retrovirus particles in the vicinity of the stem cells over that
which is obtained in
the absence of such means, so that the chance of infection of the stem cells
is enhanced. The
recombinant retrovirus contains genetic information to be introduced into the
hematopoietic
stem cells and has a host range which includes primate hematopoietic stem
cells. In some cases
3o it is preferred that the isolated hematopoietic cell population is enriched
for hematopoietic stem
cells before the hematopoietic stem cells are brought in close physical
contact with the
recombinant retrovirus. It is preferred that the genome of the recombinant
retrovirus is based on
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a retroviral vector which is derived fiom a viral MuLV vector. It is
furthermore preferred that
the recombinant retrovirus has an amphotropic host range. According to the
invention the close
physical contact provides m efficient genetic modification of the primate
hematopoietic stem
cells. The close physical contact can be accomplished by various means, which
are exemplified
in the different embodiments of the invention. Those skilled in the art will
be able to use other
means to achieve said close physical contact without departing from the
present invention.
The term "hematopoietic stem cell" is understood to mean a cell that has the
following
characteristics: ( 1 ) it has thn ability t:o differentiate into any type of
cell of the blood cell system,
and (2) it has the capacity tn multiply itself without losing its
characteristics 1 and 2. The term
~o "hematopoietic cell" is understood t~o mean any cell of the blood cell
system, independent of its
lineage commitment or maturation state. Thus, "hematopoietic cells" include
"hematopoietic
stem cells". The term "primates" is understood to mean all primates, including
man. Preferably,
the gene therapy concerns man. By "close physical contact" is intended a
contact which
enhances the local concenti~ation of retrovirus particles in the direct
vicinity of the target cell
t 5 beyond that obtained under standard conditions, where "standard
conditions" are those where
retrovirus particles and tarl;et cells ~~re mixed together in a liquid
solution at normal gravitation.
In one embodiment of the invention said isolated hematopoietic cells from a
primate are,
by means of a cocultivation, exposed to cells that produce the recombinant
retrovirus. During
this cocultivation said isolated hematopoietic cells are in the direct
vicinity of said virus-
20 producing cells. In particular, the hematopoietic stem cells from primates
are subject to close
contact and these cells adhere, in part or possibly preferentially, to said
virus-producing cells.
During the cocultivation said virus-producing cells continuously produce new
recombinant
retroviruses that are shed firom the cell membrane into the culture medium.
After their
production, said recombin~uut retroviruses have a limited life span that
depends at least in part on
25 . their nature, on the culture temperature and on the composition of the
culture medium. Hence,
the shorter the distance said recombinant retroviruses have to travel from the
site where they
were shed into the mediurr~ towards the isolated hematopoietic cells from a
primate the higher is
the chance for a successful genetic modification of said isolated
hematopoietic cells of a
primate. in this aspect of the invention, therefore, the most ef~xcient
genetic modification is
30 obtained for the subset of raid isolated hematopoietic cells from a primate
that most intimately
adhere to said virus-produ~;,ing cells. For this reason, it is preferred that
following cocultivation
both non-adherent and adr~erent cells are harvested.
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In another embodiment of the invention the intimate interaction between said
virus-
producing cells and said hematopoietic stem cells is further improved by
forcing these cells
together. This can be accomplished by various physical means, including but
not restricted to
increasing the gravitational force to enhance sedimentation of the
hematopoietic stem cells onto
the virus-producing cells by centrifugation, centrifuging a mixture of both
cell populations onto
a solid material, concentrating said mixture on the same physical site by
electrodif~usion,
forcing by pressure or centrifugation said mixture onto a porous solid
material with pores large
enough in size to allow passage of the fluid medium but small enough in size
to prevent passage
of said mixture. In the latter application of the invention, said pressure is
either positive
t o pressure applied to said fluid medium or negative pressure applied to said
porous solid material
or to a space past said porous solid material. Alternatively, said intimate
interaction can also be
improved by performing the culture in the presence of a compound that binds
both the virus-
producing cells and the hematopoietic stem cells.
In yet another embodiment of the invention said hematopoietic stem cells are
cultured in
recombinant retrovirus containing medium in the presence of a compound that
binds both the
recombinant retrovirus and the hematopoietic stem cell, thus providing the
close physical
contact between said hematopoietic stem cell and said recombinant retrovirus.
Said compound
is characterized by its capacity to bind ( 1 ) said hematopoietic stem cell,
and (2) said
recombinant retrovirus and/or said virus-producing cell. It is preferred that
said compound
zo besides binding to said recombinant retrovirus or virus-producing cell
preferentially, ors even
exclusively, binds to said hematopoietic stem cell. 1n this way, said compound
selectively
increases the genetic modification of said hematopoietic stem cell. Said
compound comprises
one or more molecules that are selected from or are derived from synthetic or
naturally
occurring molecules including but not restricted to polymers, antibodies,
peptides, cell surface
membranes or fragments or components thereof, extracellular matrices or
components thereof,
intact cells, and complete tissues or components thereof. Said molecules
include composite
molecules containing parts from molecules of different origin. Preferred
compounds in the
invention are derived from or are components of the natural hematopoietic
microenvironment
present in the bone marrow of animals. Said hematopoietic stem cells by nature
closely interact
3o with cells and extracellular matrix molecules present in said hematopoietic
microenvironment. .
In addition, said cells produce cytokines that support the maintenance and
functioning of said
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hematopoietic stem cells a~zd said e:Ktracellular matrix molecules bind
cytokines that support the
maintenance and functionvng of said hematopoietic stem cells.
The method of the invention is also performed using a different kind of
compound that
( 1 ) binds to said recombinant retrovirus vector and (2) is immobilized on a
solid support
material. Said hematopoietic cells of a primate are brought in close contact
with said solid
support material (and there;by with ;said bound recombinant retrovirus vector)
by any other
means exemplified in the various embodiments of the invention (like gravity,
electrodiffusion,
and fluid flow).
In another embodiment of the invention the close contact between the
recombinant
t o retrovirus and the hematopoietic stem cell is accomplished by forcing said
recombinant
retrovirus towards said hematopoietic stem cell by any of various physical
means. These
include but are not restricted to increasing the gravitational force by
centrifugation to induce
settling of the recombinant retrovirus onto the hematopoietic stem cell,
causing the recombinant
retrovirus to move toward; the hematopoietic stem cell by electrodif~usion,
and forcing the
t s recombinant retrovirus-containing :medium through a bed of hematopoietic
cells including said
hematopoietic stem cell. In the lather case, said hematopoietic cells are
seeded on top of a
porous solid material with pores large enough in size to allow passage of the
fluid medium but
small enough to prevent s~iid cells oo pass. In this application of the
invention, said force is
provided by either normal gravity, increased gravity through centrifugation,
positive pressure
2o applied to said medium, or negative pressure applied to said porous solid
material or toy a space
past said porous solid material. In this aspect of the invention it is
preferred but not essential
that the solid material use~3 binds the recombinant retrovirus.
As is clear from th.e above, the invention provides means to bring isolated
hematopoietic
cells including hematopoietic stem cells from a primate in close physical
contact with a
25 recombinant retrovirus. 'Ibis is accomplished either directly, by bringing
the recombinant
retrovirus itself in close proximity of said isolated hematopoietic cells, or
indirectly, by bringing
cells that produce the recombinant retrovirus in close proximity of said
isolated hematopoietic
cells.
According to the invention, it is preferred that the retroviral vector
comprises two LTRs
30 (long terc~inal repeats) derived from a viral MuLV vector and the 5' part
of the gag gene of a
MuLV. 'Ihe MuLV sequences are preferably derived from the viral Mo-MuLV vector
(Moloney
Murine Leukemia Virus), while at least the 3' -LTR is a hybrid LTR which
contains the PyF101
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enhancer instead of the Mo-MuLV enhancer. To this end, preferably the
retroviral vector
pLgXL(OMo+pyF101) is used, wherein X represents the genetic information to be
introduced
into the bone marrow cells.
According to the invention, producer cells that can be used include all
recombinant
s retroviral vector producing cell lines with a host range that includes
primates. Several examples
of producer cell lines that produce retroviral vectors with the
LgXL(OMo+pyF101) structure
useful in the invention have been disclosed in Patent Application W096/35798.
The cells that
produce the recombinant retrovirus are preferably recombinant mammalian cells
which contain
and express the gag, pol and env genes of MuLV. The env gene is preferably
derived from an
t o amphotropic MuLV. The gag, pol and env genes of MuLV in the recombinant
mammalian
cells are preferably distributed over at least two different eukaryotic
expression vectors.
Further, it is preferred that each packaging construct is associated with a
selectable marker gene.
As recombinant mammalian cells GP+envAMl2 cells are used, it is further
preferred that the
cells that produce a recombinant retrovirus contain several copies of the
retroviral vector.
According to the invention, it is further preferred that the cultivation of
hematopoietic
stem cells in recombinant retrovirus supernatant or with cells that produce
recombinant
retrovirus occurs in the presence of serum and at least one hematopoietic
growth factor. In some
embodiments of the invention, it is further preferred to culture said
hematopoietic stem cells for
a period of time in the absence of recombinant retrovirus and virus-producing
cells before being
2o subjected to genetic modification with recombinant retrovirus or to culture
said hematapoietic
stem cells for a period of time in the absence of recombinant retrovirus and
virus-producing
cells after having been subjected to genetic modification with recombinant
retrovirus.
The invention further provide cells that produce a recombinant amphotropic
retrovirus
with a genome based on a retroviral vector, preferably one which is derived
from a viral MuLV
vector, which contains genetic information that is suitable to be introduced
into bone marrow
cells of a primate according to the method described herein.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a method for introducing a gene X into isolated
hematopoietic
3o cells including hematopoietic stem cells from a primate, whereby said
isolated hematopoietic
cells are brought in close contact with a recombinant retrovirus. Preferably,
said recombinant
retrovirus is an amphotropic retrovirus whose genome is composed of the
recombinant retroviral
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11
vector pLgXL(aMo+PyF 101 ) wherein gene X represents a nucleic acid molecule
inserted
therein that encodes a ribonucleic acid molecule or a protein which is of
importance for gene
therapy. The invention is comprised of a number of useful components: a
recombinant
retroviral vector pLgXL(OMo+PyF 11~ i ), a virus-producing cell line shedding
recombinant
pLgXL(~Mo+PyF 1 O 1 ) retrovirus, anal a method by which isolated
hematopoietic cells or
purified hematopoietic stem cells of a primate are provided with gene X.
Hematopoietic cells
Many different standard procedures are known in the art for the collection,
storage,
t o processing, and reinfusion of haemopoietic cells from bone marrow,
peripheral blood, fetal
liver, or umbilical cord blood of primates, as well as for conditioning of the
recipient and for
post-transplantation supporl:ive care (see, e.g., Bone Marrow and Stem Cell
Processing: A
Manual of Current Techniques, (1992) eds. E.M. Areman, H.J. Deep, and R.A.
Sacher, F.A.
Davis Company, Philadelphia, pp. 487; Marrow Transplantation: Practical and
Technical
15 Aspects of Stem Cell Reconstitution, ( 1992) eds. R. A. Sacher and J. P.
AuBuchon, American
Association of Blood Bank;, Bethesda, MD, pp. 187). Several methods for stem
cell
enrichment by CD34+ cell selection are known in the art that use commercially
available
materials. They have been compared by Wynter et al., Stem Cells (1995) 13: 524-
532. The
MACS Cell Sorting method (Miltenyi Biotec, Germany) gives the best results
with respect to
2o purity and recovery, and is thus preferred.
True in vitro tests for haemopoietic stem cells do not exist, but phenotypic
analysis is
usually performed as an indicator of the quality of both the isolated material
and the graft after
gene transfer (Knaan-Shan:cer et al., Gene Therapy (1996) 3:323-333). In the
experiments with
rhesus monkeys described iin the examples this was not done, because not all
essential
25 antibodies for this analysis react with rhesus monkey cells. There is no
special treatment of the
cells prior to cultivation with retrovirus particles.
Recombinant retroviral vector L (OMo+PyF 1 O 1 )
The recombinant re,trovirai vector includes DNA elements originating from a
MuLV
3o which are'necessary in cis 'for the p~~ckaging, reverse transcription and
integration of the
retroviral genome; these include two so-called Long Terminal Repeats (LTR) and
the so-called
packaging sequences. In the LTR a modification has been provided by replacing
the enhancer
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12
originating from MuLV with the enhancer of the polyoma virus strain PyF 101
(Linney et al.,
( 1984) Nature 308:470). In the plasmid construct, it is not necessary that
this modification is
present in both LTRs; only the 3' LTR must be provided with the modification
since that portion
of the LTR ends up in both LTRs after a viral infection (Van Beuschem et al.,
(1990) J. Exp.
Med. 172:729; and Valerio et al., (1989) Gene 84:419), and the 5' part of the
MuLV gag-
encoding sequences such as present in the vector N2 (Armentano, D., S. F. Yu,
P. W. Kantoff,
T. Von Ruden, W. F. Anderson and E. Gilboa, (1987), Effect of internal viral
sequences on the
utility of retroviral vectors, J. Virol. 61:1647), so as to effect a higher
viral titer. Optionally, the
ATG initiation codon of gag can be mutated by means of site-directed
mutagenesis, so that it is
1 o no longer a translation start site. The only absolute requirements for the
vector are (i) the
inclusion of DNA elements necessary in cis for the packaging, reverse
transcription, and
integration of the retroviral genome, and (ii) that the gene X be placed
within a proper
transcription unit, wherein it is preferred that this transcription unit is a
natural viral
transcription unit (no internal promoter). The pLgXL(OMo+pyF 1 O 1 ) vector
meets these
~ 5 requirements and includes some further improvements, as exemplified above.
The retroviral vector is included in a plasmid construct having plasmid
sequences
necessary for propagation of the vector in E. Coli bacteria such as for
instance pBR322 (Bolivar
et al., (1977) Gene 2:95) or a vector from the pUC series (Vieira and Messing,
(1982) Gene
19:259); on these, both an origin of replication and a selectable gene (for
instance for ampicillin
20 of tetracycline resistance) are present, together with gene X. The term
"gene" is to be
understood to mean a nucleic acid molecule encoding a ribonucleic acid
molecule or protein. It
includes naturally occurring nucleic acid molecules and synthetic derivatives
thereof. Useful
genes that encode a ribonucleic acid molecule or a protein which is of
importance for gene
therapy include, but are not restricted to, all genes associated with
hereditary disorders wherein a
25 therapeutic effect can be achieved by introducing an intact version of the
gene into somatic cells.
Most of them are documented in:
McKusick, Mendelian Inheritance in Man, Catalogs ofAutosomal
Dominant,Aautosomal
Recessive, and X Linked Phenotypes. Eighth edition. John Hopkins University
Press ( 1988),
and Stanbury et al., The Metabolic Basis of Inherited Disease. Fifth edition.
McGraw-Hill
30 (1983).
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Examples of gene X include:
genes associated with diseases of the carbohydrate metabolism such as for:
fivctose-1-
phosphate aldolase; fructo~~e-1,6-diphosphatase; glucose-6-phosphatase;
lysosomal a-1,4-
giucosidase; amylo-1,6-glucosidase; amylo-(1,4:1,6)-transglucosidase; muscular
phosphorylase;
s liver phosphorylase; muscular phosphofructokinase; phosphorylase-b-kinase;
galactose-1-
phosphate uridyl transferase; galaci:okinase; all enzymes of the pyruvate
dehydrogenase
complex; pyruvate carbox;ylase; 2-oxoglutarate glyoxylate carboligase; and D-
glycerate
dehydrogenase;
genes associated with diseases of the amino acid metabolism such as for:
phenylalanine
to hydroxylase; dihydrobiopterin synfhetase; tyrosine aminotransferase;
i:yrosinase; histidase;
fiunarylacetoacetase; gluta~thione synthetase; y-glutamylcysteine synthetase;
orinithine- 8-
aminotransferase; carbamoylphosphate synthetase; ornithine
carbamyltransferase;
argininosliccinate syntheta~se; argininosuccinate lyase; arginase; L-lysine
dehydrogenase; L-
lysine ketoglutarate reduct:ase; valine transaminase; leucine isoleucine
transaminase; "branched
15 chain" 2-keto acid decasboxylase; i.sovaleryl CoA dehydrogenase; acyl-CoA
dehydrogenase; 3-
hydroxy-3-methylglutaryl CoA lyase; acetoacetyl CoA 3-ketothiolase; propionyl
CoA
carboxylase; methylmalonyl CoA mutase; ATP:cobalamine adenosyltxansferase;
dihydrofolate
reductase; methylene tetrahydrofolate reductase; cystathionine (3-synthase;
sarcosine
dehydrogenase complex; proteins belonging to the glycine cleavage system; ~3-
alanine
2o transaminase; serum carnosinase; and cerebral homocarnosinase;
genes associated v~rith diseases of fat and fatty acid metabolism such as for:
lipoprotein
lipase; apolipoprotein C-If; apolipoprotein E; other apolipoproteins; lecithin
cholesterol
acyltransferase; LDL receptor; liver sterol hydroxylase; and "Phytanic acid" a-
hydroxylase;
genes associated v~rith lysosomal defects such as for: lysosomal a-L-
iduronidase;
25 lysosomal iduronate sulfa~tase; lysosomal heparin N-sulfatase; lysosomai N-
acetyl-a-D-
sulfai:ase; lysosomal acetyl CoA:a-glucosaminide N-acetyltransferase;
lysosomal N-acetyl-a-D-
glucosaminide 6-sulphatase; lysosomal galactosamine 6-sulphate suifatase;
lysosomal ~i-
galactosidase; lysosomal arylsulfatase B; lysosomal ~3-giucuronidase; N-
acetylglucosaminylphospllotxansfe:case; lysosomal a-D-mannosidase; lysosomal a-
3o neuramirudase; lysosomall aspartylglycosaminidase; lysosomal a-L-
fucosidase; lysosomal acid
lipase; lysosomal acid ceramidase; lysosomal sphingomyelinase; lysosomai
glucocerebrosidase;
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lysosomal galactosylceramidase; lysosomal arylsulfatase A; a-galactosidase A;
lysosomal acid
(3-galactosidase; and a-chain of the lysosomal hexosaminidase A;
genes associated with diseases of the steroid metabolism such as for: 21-
hydroxylase;
11 ~i-hydroxylase; androgen receptor; steroid Sa-reductase; steroid sulfatase;
genes associated with diseases of the purine and pyrimidine metabolism such as
for:
phosphoribosylpyrophosphate synthetase; hypoxanthine guanine
phosphoribosyltransferase;
adenine phosphoribosyltransferase; adenosine deaminase; purine nucleoside
phosphorylase;
AMP deaminase; xanthine oxidase; orotate phosphoribosyltransferase; orotidine
5'-phosphate
decarboxylase; and DNA repair enzymes;
1 o genes associated with diseases of the porphyrin and heme metabolism such
as for:
uroporphyrinogene III cosynthase; ferrochelatase; gorphobilinogene deaminase;
coproporphyrinogene oxidase; proporphyrinogene oxidase; uroporphyrinogene III
synthase;
uroporphyrinogene decarboxylase; bilirubin UDP-glucuronyltransferase; and
catalase.
genes associated with diseases of the connective tissue, muscles and bone such
as for:
15 lysyl hydroxyiase; procollagen peptidase; al-antitrypsin; dystrophin;
alkaline phosphatase; and
guanosine nucleotide regulatory protein of the adenyl cyclase complex;
genes associated with diseases of the blood and blood-forming organs such as
for: blood
coagulation factor V; blood coagulation factor VII; blood coagulation factor
VII; blood
coagulation factor IX; blood coagulation factor X; blood coagulation factor
XII; blood
2o coagulation factor XIII; all other blood coagulation factors; all genes
associated with
osteopetrosis such as for: "carbonic anhydrase II"; thrombocytes membrane
glycoprotein Ib;
thrombocytes membrane glycoprotein IIb-)TIa; spectrin; pyruvate kinase;
glucose-6-phosphate
dehydrogenase; NADH cytochrome b5 reductase; (3-globin; and a-globin;
genes associated with diseases of transport systems such as for: lactase;
sucrase-a-
25 dextrinase; 25-hydroxyvitamin D3-1-hydroxylase; and cystic fibrosis
transport regulator;
genes associated with congenital immunodeficiencies such as for: the proteins
of the
complement system including B, Clq, Clr, C2, C3, C4, C5, C7, C8 and C 10; the
inhibitor of C1,
a component of the complement system; the inactivator of C3b, a component of
the complement
system;
3o genes for X-linked immunodeficiencies such as for: one of the enzymes of
the NADPH
oxidase complex; myeloperoxidase; and the syndrome of Wiscott Aldrich and
Ataxia
Telangiectasia;
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genes coding for hormones as well as the genes coding for their receptors such
as for
instance for growth hormone.
Gene X also include;s genes ~~rhich (to date) have not been associated with a
hereditary
defect but with which gene therapy c;an be practiced in some manner. These
include: the gene
5 for tyrosine hydroxylase, ding resistance genes such as for instance: the P-
glycoprotein P 170
(the so-called mufti drug resistance gene mdrl); mdr 3; dihydrofolate
reductase (DHFR) and
methotrexate resistant isotypes thereof; metallothionine; aldehyde
dehydrogenase (ALDH); and
glutathione transferase; genes coding for all cytokines including for instance
all interieukins and
all interferons; genes coding for all ~~owth factors; genes coding for all
growth factor receptors;
to genes coding for all transplantation .antigens such as for instance the
major and minor
histocompatibility antigens; genes capable of affording resistance against
infectious organisms,
such as for instance TAR decoys (Siallenger et al., (1990) Cell 63:601),
antisense ribonucleic
acid molecules, ribozymes, and intracellular antibodies; genes of infectious
organisms which
can be used for vaccination. purposes such as for instance the envelope gene
of HIV; and genes
15 which can be used for negative selection such as for instance the thymidine
kinase gene of the
Herpes simplex virus against which selection can be effected with substrates
such as for instance
gancyclovir or acyclovir (H~orelli et al., ( 1988), Proc. Natl. Acad Sci. USA
85:7572; and
Mansour et al., (1988) Nature 336:,48).
2o The virus=producing cells i
In order to obtain a stable, selectable virus-producing cell line which
produces the
amphotropic recombinant retroviru:~, pLgXL(~Mo+pyF 1 O 1 ) is introduced into
an amphotropic
packaging cell Iine that is ~~elected for the presence of the DNA sequences
which are of
importance for the production of the viral proteins. One example of such a
cell line is
GP+envAml2 (Markowitz et al., (1988) Virology 167:400). It has been
demonstrated, on the
other hand, that yrCRIP is :not selectable and is unstable with respect to
virus production (Danos
and Mulligan, ( 1988) Proc. Natl. A~cad Sci. USA 85:6460).
The selectable packaging cell line is based on mammalian cells and produces
all viral
proteins that are coded by the gag, ivol and env genes of MuLV. The env gene
must originate
3o from a vilvs with a tropism includuig primates, and is preferably derived
from an amphotropic
MuLV. In order to obtain expression of the aforementioned viral genes, they,
while cloned in a
eukaryotic expression vector, must be under control of a promoter active in
the host, preferably
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a RNA polymerise II promoter, and be followed by a polyadenylation signal. On
these so-called
packaging constructs, all three viral genes may be present simultaneously as
for instance
described by Miller (Miller and Buttimore (1986) Mol. Cell. Biol. 6:2895), but
the genes may
also occur separately on two expression vectors as described by Markowitz
(Markowitz et al.,
s ( 1988) Virology 167:400). 'This last is to be preferred because it reduces
the chances of
recombination events leading to helper virus formation.
As stated, a useful characteristic of the packaging cell line to be used for
this invention is
the possibility it provides meansof selecting for the presence of the above-
mentioned packaging
constructs. This can be achieved by effecting a physical association of the
packaging constructs
i o with a selectable marker gene. This association can be achieved by
combining them in one
vector (as done with pGag-PoIGPT in reference (Markowitz et al., (1988) J.
Virol. 62:1120) or
by means of a so-called cotransfection (review in for instance (Pellicer et
al., ( 1980) Science
209:1414). The successfully transfected cells can then be isolated by
selecting for the marker
gene. Since the cotransfected DNA fragments mostly end up ligated to each
other at one place
15 in the genome of the transfected cell (Pellicer et al., ( 1980) Science
209:1414), the thus selected
cells will mostly contain the packaging construct as well. In view of the fact
that yCRIP cells
have been made in this way and, nevertheless, are not selectable, the last
procedure is not always
successful and the construction of vectors with the marker gene cloned into it
is to be preferred.
As a marker gene, genes coding for a large number of different proteins can be
used.
2o Widely used and preferred marker genes are: the neomycin resistance gene
(Southern and Berg,
{1982) J. Mol. Appl. Genet. 1:327), the hygromycin resistance gene
(Blochlinger and Diggelman
(1984) Mol. Cell. Biol. 4:2929), the E. coli xanthine-guanine phosphoribosyl
transferase (gpt)
gene (Mulligan and Berg (1980) Science 209:1422), the histidinol gene (Hartman
and Mulligan
( 1988) Proc. Natl. Acid Sci. USA 85:8047), the herpes simplex virus thymidine
kinase gene
25 (Colbere-Garapin et al., (1979) Proc. Natl. Acid Sci. USA 76:3755) and the
methotrexate
resistant isotype of dihydrofolate reductase (Simonsen and Levinson (1983)
Proc. Natl. Acid
Sci. USA 80:2494). These genes must also be under control of a suitable
promoter, in particular
a RNA polymerise II promoter, and be followed by a polyadenylation signal.
The introduction of pLgXL(OMo+PyF101 ) can be effected by means of various
physical
3o techniques such as calcium-phosphate precipitation, electroporation or
lipofection (Graham and
Van der Eb, ( 1973) Nucl. Acids Res. 15:1311; Potter et al., ( 1984) Proc.
Natl. Acid. Sci. USA
81:7161; Felgner and Ringold (1989) Nature 337:387; Felgner et al., (1987)
Proc. Natl. Acid.
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Sci. USA 84:7413). If the packaging cells cannot be selected for the presence
of
pLgXL(~Mo+PyF101), use is made of a selectable marker such as for instance an
expression
vector of the neomycin resistance gene which is transfected together with
pLgXL{OMo+PyF101). Th.e successfully transfected cells are then be selected by
selecting for
s the marker gene. Since the DNA fragments mostly end up ligated to each other
in one place in
the genome of the transfect~ed cell, the thus selected cells will mostly
contain the retroviral
vector as well.
A preferred procedl~re is the introduction of pLgXL(AMo+PyF 1 O 1 ) via an
infection.
Since viruses are not capable of infecting packaging cells of the same
tropism, use must be
t o made of a version of the recombinant retrovirus with a different tropism
which is obtained by
introducing the DNA initially via a physical technique into packaging cells
with said different
tropism. For example, ecotxopic virus produced by ecotropic packaging cells
transfected with a
pLgXL(~lVlo+pyF101) construct can be used to infect amphotropic packaging
cells. The
infected cells are cloned and then tested for their ability to produce virus.
15 Further, it is possible to obtain cell lines producing a higher titer of
the virus by
introducing several copies ~of the retroviral vector into the packaging cells
using the so-called
"ping-pong" method (Beshwick et a~!., (1988) Proc. Natl. Acad. Sci. USA
85:5404; and Kozak
and Kabat ( 1990) J. Virol. 64:3500). In this method, an eeotropic virus-
producing cell line is
cocultivated with amphotropic packaging cells, which can give rise to repeated
infections. In
20 order to enable the amphotropic cells to be cloned back after this
cocultivation, they mug st be
selectable with selective media in which the ecotropic packaging cells do not
survive. By
plating the cells in such medium, the proper virus-producing clones can be
isolated and
subsequently analyzed for their capacity to produce the recombinant virus.
25 Method by which hematopoietic cells of a primate can be provided with sene
X, in such a
manner that the re eng eraticfn capacity of the hematonoietic cells is
maintained and autolo~~ous
transplantation of the hematopoieti<; cells gives rise to a >;eneticallv
modified hematopoietic
s s~ tem
The above-mentioned recombinant retroviral vectors are used for the efficient
3o introductibn of gene X into hematopoietic cells of primates by bringing
said hematopoietic cells
in close physical contact with said recombinant retroviral vectors. A key
aspect of the invention
is the realization that the efficiency of gene transfer is in part dependent
on the chance for a
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recombinant retroviral vector particle to associate with its receptor on the
surface of a
hematopoietic target cell. Thus, important factors determining the efficiency
of recombinant
retroviral vector-mediated gene transfer into hematopoietic cells of primates
include ( 1 ) the
concentration of recombinant retroviral vector particles at the site of the
hematopoietic target
cell, (2) the density and affinity of receptors for the recombinant retroviral
vector on the surface
of the target cell, (3) the stability of the recombinant retroviral vector
particle, and (4) the
stability of the hematopoietic target cell. Information relative to these
factors is as follows.
To optimize the concentration of recombinant retroviral vector particles at
the site of the
target cell, one can increase the total concentration of recombinant
retroviral vector particles in
1 o the culture medium, by improving the virus-producing cell line or the
virus production and
harvest procedure. This invention provides an additional method to increase
the concentration
of recombinant retroviral vector particles at the site of the hematopoietic
target cell, i.e., by
establishing a close physical contact between the recombinant retroviral
vector and said target
cell according to one of several means which are exemplified in detail below.
For the scope of the invention, the density and affinity of receptors for the
recombinant
retroviral vector on the surface of a certain target cell are regarded as
naturally constant factors.
Although perhaps receptor expression or integrity on target cells could be
influenced, e.g. by
changing the culture conditions, this is not part of the invention. It is
realized, however, that
receptor densities may intrinsically differ between different cell types
significantly. It is:
furthermore realized that especially for cell types with very few functional
receptors fot the
recombinant retroviral vector, which may include the hematopoietic stem cell,
it is important to
enhance the chance for a virus-to-cell encounter. This will be even more
important when a
target cell with few functional receptors is part of a cell mixture containing
other cell types
having higher functional receptor densities.
In general, the half life of infectious recombinant retroviral vector
particles under
standard culture conditions is low (3-9 hours; e.g., Kotani et al., Hum. Gene
Ther. 5( 1994) 19-
28; Forestell, et al., Gene Ther. 2(1995) 723-730). This half life can be
increased by lowering
the culture temperature to 32°C (Kotani et al., Hum. Gene Ther. 5(
1994) 19-28). Methods to
increased the stability of the recombinant retroviral vector particles are not
part of the invention.
3o It is reali~d, however, that the invention providing an increased chance
for a virus-to-cell
encounter is of particular importance for retroviral vectors with a short half
life.
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It is of critical importance that a method to transfer a gene X into a certain
target cell
allows said target cell to regain all of its characteristics. Especially in
the case of hematopoietic
stem cells of primates this has previously been difficult, if not impossible,
to achieve. Gene
transfer procedures tested c~n bone marrow grafts of primates led to a
dramatic loss of the in vivo
regenerating capacity of the; grafts (y ee above). The present invention
provides a method for
efficient transfer of gene X into hematopoietic stem cells of primates that
does not significantly
affect the in vivo regenerating capacity of the manipulated graft.
Numerous different procedures for harvest, processing, storage, shipping, etc.
of human
haemopoietic cells are in u:~e and known to those of skill in the art. Various
means of bringing
t o together of the recombinant retrovirus particles and hematopoietic target
cells.
General procedure for recombinant retroviral-vector mediated gene transfer
into hematopoietic
cells of primates:
The hematopoietic .cells of a primate are suspended in a suitable culture
medium for
hematopoietic cells containung recombinant retrovirus vector particles. Said
hematopoietic cells
are either total mononuclear cells or are cell populations that are enriched
for stem cells
according to various methods known in the art, including but not restricted
to, density separation
(Percoll, BSA) and positim: selection for CD34+ cells (FACS sorting, Dynabeads
imununoselection, Miltenyi MACS selection, AIS CELLector flask selection, or
CellPro
zo CEPRATE selection) or depletion of cells carrying mature cell type cell
surface markerrs. Many
different suitable culture media are commercially available. They include, but
are not restricted
to DMEM, )1V~M, and a-1!VIEM, with 5-30% serum and often further supplemented
with, e.g.,
BSA, one or more antibiotiics. L-glutamine, 2-mercaptoethanol, hydrocortisone,
and
hematopoietic growth factors. Recombinant retrovirus vector particles are
harvested into this
medium by incubating virus-producing cells in this medium. To enhance gene
transfer, usually
compounds such as polybrene, protamine sulphate, or protamine HC 1 are added.
Usually, the
cultures are maintained for 2-4 day:. and the recombinant retrovirus vector
containing medium is
refreshed daily. Optionall~~, the hernatopoietic cells are precultured in
medium with growth
factors but without recombinant retrovirus vector particles for up to 2 days,
before adding the
recombinant retrovirus vector containing medium.
For successful gene; transfer it is essential that the target cells undergo
replication in
culture (without differentiation). Most harvested haemopoietic stem cells are
resting cells.
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Therefore, a stimulus to enter cell cycle during culture is needed. This can
be accomplished by
adding recombinant haemopoietic growth factors (HGF), including different
combinations of
HGF such as interleukin-3, interleukin-6, and steel factor (SCF). In cultures
with stromal cell
support, or other cells which produce the necessary HGF, such as a retrovirus
producing cell
line, HGF addition is not needed.
The method of the invention is performed according to one of the following
procedures,
either indirectly, by bringing hematopoietic cells of a primate in close
physical contact with
virus-producing cells, or directly, by bringing hematopoietic cells of a
primate in close physical
contact with recombinant retroviral vector particles.
1 o The following are examples of indirect methods which are used.
i) Cocultivation of hematopoietic cells of a primate with virus producing
cells.
Said virus-producing cells and the hematopoietic cells from a primate are
mixed at the initiation
of the coculture, or a monolayer of adherent virus-producing cells is
established before adding
said hematopoietic cells from a primate. Said virus-producing cells may have
been damaged
15 prior to initiation of the coculture by, e.g., a lethal dose of
irradiation, but can be used so long as
they continue to shed recombinant retroviral vector particles into the medium.
Said virus
producing cells have the capacity to bind said hematopoietic cells to their
surface. Virvs-
producing cells based on the commonly used packaging cells derived from mouse
fibroblasts
have this capacity. Due to the intimate interaction between said virus-
producing cell and said
2o hematopoietic cell the recombinant retroviral vectors produced by said
virus-producing cell only
have to travel a very short distance to reach said hematopoietic cell. It may
even occur that a
recombinant retroviral vector fuses with the membrane of the hematopoietic
cell while being
shed from the membrane of the virus-producing cell. The invention thus
provides a transduction
method that minimizes the time during which the recombinant retroviral vector
is exposed to de-
stabilizing components of the environment. The most efficient genetic
modification is obtained
for the subset of hematopoietic cells of a primate that most intimately adhere
to the virus-
producing cells. It is preferred that said virus-producing cells
preferentially, or even
exclusively, bind hematopoietic stem cells. In this way, the genetic
modification of said
hematopoietic stem cells is selectively increased. In this embodiment of the
invention, it is
3o preferred Shat the cocultivation takes place for three to four days in the
presence of serum and
one or more hematopoietic growth factors such as for instance interleukin 3
(II,-3). Following
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cocultivation, both the non-adherent and the adherent cells are harvested from
the culture (the
last-mentioned cells can be obtained by, e.g., trypsinization) and used as the
transplant.
ii). Cocultivation of hematopoietic cells of a primate with virus-Qroducins
cells at
increased gravitational force. This .embodiment of the invention provides a
further improvement
s of and includes the advanW ges of the procedure described under (i). Apart
from the increased
gravitational force, the procedure is performed as described under (i). By
increasing the
gravitational force two effc;cts are being accomplished, i.e., (1) the
intimate interaction between
the virus-producing cells and the hematopoietic cells from a primate is
further enhanced, and (2)
recombinant retrovirus vector particles that have been shed into the culture
medium are
1 o prevented from traveling away from the hematopoietic cells, thus
increasing the local
concentration of said particles. Said increased gravitational force is
achieved by performing the
cocultivation while spinnvig the container with the culture around an axis of
rotation. Said axis
can intersect said container or be located outside of said container. Useful
centrifuges to spin
the cultures according to tlae invention are known in the art. The
gravitational force is
t s maximized, but should not exceed the maximal gravitational force that
allows functional
survival of the virus-producing cellls, the recombinant retroviral vector
particles and the
hematopoietic cells from a primate. Usually, said gravitational force will not
exceed 2500 g.
As a result of the further uncreased gene transfer efficiency obtained with
this embodiment of the
invention, the coculture duration can be significantly shortened. Usually,
this procedure will not
2o be performed for more thaw eight consecutive hours.
iii). Cocultivati~on of hematopoietic cells of a primate with virus-producing
cells with
increased inter-cellular contact accomplished by electrodiffusion. This
embodiment of the
invention provides an alternative improvement of and includes the advantages
of the procedure
described under (i). Apart from the electxodiffusion, the procedure is
performed as described
2s under (i). Because the hematopoie;tic cells of a primate, the virus-
producing cells, and the
recombinant retroviral vector particle are all negatively charged they can be
forced to move
towards a positive electrode. By performing the cocultivation procedure in an
electrophoresis
unit said negatively charged cells and vectors are concentrated. This way, two
objectives are
accomplished, i.e., ( 1 ) the; intimate interaction between the virus-
producing cells and the
3o hematopo~etic cells from a primate is further enhanced, and (2) the
recombinant retrovirus
vector particles that have been shed into the culture medium are prevented
from traveling away
from the hematopoietic cells, thus increasing the local concentration of said
particles.
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Electrophoresis units useful for this aspect of the invention are known in the
art. Said
electrophoresis unit preferably contains two chambers separated by a semi-
permeable
membrane, with pore sizes that do not permit passage of said cells and
vectors. In such a two-
chamber electrophoresis unit said cocultivation is performed in the chamber
containing the
negative electrode. The voltage applied between the electrodes is maximized,
but kept below a
value that causes significant damage to said hematopoietic cells of a primate,
said virus-
producing cells, and said recombinant retroviral vector particles. To further
reduce said
damage, said voltage may be applied periodically. Also in this embodiment of
the invention,
said virus-producing cells and said hematopoietic cells from a primate are
mixed at the initiation
of the coculture, or a monolayer of adherent virus-producing cells is
established on the surface
of said semi-permeable membrane before adding said hematopoietic cells from a
primate.
iv). Cocultivation of hematopoietic cells of a primate with virus-producin,~
cells with
increased inter-cellular contact accomplished by fluid flow. This embodiment
of the invention
provides another alternative improvement of an includes the advantages of the
procedure
t 5 described under (i). Apart from the fluid flow, the procedure is performed
as described under
(i). Said fluid flow is brought about by forcing the culture medium through a
porous solid
material with pores large enough in size to allow passage of said culture
medium but small
enough in size to prevent passage of said hematopoietic cells of a primate and
said virus-
producing cells. Said pores may or may not allow passage of the recombinant
retroviral vectors.
2o The force driving said fluid flow is exercised by normal or increased
gravitational force or by
positive pressure applied to said culture medium or by negative pressure
applied to said porous
solid material or to a space past said porous solid material. Said increased
gravitational force is
achieved by performing the cocultivation while spinning the container with the
culture around
an axis of rotation. Said axis can intersect said container or be located
outside of said container.
25 Said pressure is established using a pump device. Pump and centrifuge
devices useful in this
aspect of the invention are known in the art. The rate of the fluid flow
depends in part on the
size of the pores in the solid material: if said pores allow passage of the
recombinant retroviral
vectors the rate is at a value that at least compensates for random diffusion
of the recombinant
retroviral vector; if said pores do not allow passage of the recombinant
retroviral vector the rate
3o is maximized; but in no case may said rate exceed the maximal rate that
allows functional
survival of the virus-producing cells, the recombinant retroviral vector
particles and the
hematopoietic cells from a primate. Also in this embodiment of the invention,
said virus-
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23
producing cells and said he~matopoietic cells from a primate are mixed at the
initiation of the
coculture, or a monolayer of adherent virus-producing cells is established on
the surface of said
porous solid material before adding said hematopoietic cells from a primate.
v). Cocultivation of hematopoietic cells of a primate with virus-
producin~cells in
s the presence of a compound that buids both said hematopoietic cells of a
primate and said virus-
producin cg ells. This embodiment of the invention provides another
alternative improvement of
and includes the advantages of the procedure described under (i). Apart from
said compound,
the procedure is performed. as described under (i). Said compound has at least
one binding site
for said hematopoietic cell of a primate and at least one binding site for
said virus-producing
t o cell. The nature of said binding sites may be different or the same. Said
compound is a soluble
molecule or a solid support material or comprises several soluble molecules
bound directly or
indirectly to each other or comprises one or more soluble molecules bound to
the same solid
support material. Said indirect binding may be via a homogeneous or
heterogeneous complex of
molecules,' via cell surface membranes or fragments or components thereof, via
intact cells, or
15 even via a complex mixture of different cells. Said mixture of cells may be
artificially
composed or be derived from naturally occurring cell mixtures or tissues.
Thus, it is to be
understood that said comp~oundy may, e.g., comprise a complete naturally
occurring tissue.
Another nonlimiting example of a compound in this embodiment of the invention
is a tissue
culture plastic with a coating that binds to said hematopoietic cells of a
primate and said,virus-
2o producing cells. In this embodiment of the invention it is preferred that
said binding site for a
hematopoietic cell of a primate has a binding preference for hematopoietic
stem cells over other
types of hematopoietic cells.
Preferred compounds in this aspect of the invention are derived from or are
components
of the natural hematopoietic microE;nvironment present in the bone marrow of
animals. Said
25 hematopoietic stem cells b~y nature closely interact with cells and
extracellular matrix molecules
present in said hematopoie;tic microenvironment. In addition, said cells
produce cytokines that
support the maintenance and functioning of said hematopoietic stem cells and
said extracellular
matrix molecules bind cytokines that support the maintenance and functioning
of said
hematopoietic stem cells. We are using a cultured stroma cell population. This
is a complex
3o mixture of cells, extracellular matrix molecules and the cytokines produced
by the cultured
cells. The stroma culture significantly enhances the recovery of a
phenotypically defined
candidate human hematopoietic stem cell population (approx. 10-fold).
Components of the
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24
extracellular matrix include collagens, proteoglycans, fibronectin, laminin,
elastin,
glycosaminoglycans, thrombospondin, and chondronectin.
Further preferred compounds in this aspect of the invention comprise parts
that are
derived from antibodies or from peptides with a defined binding capacity. Said
peptides may be
naturally occurring or artificially synthesized or derived from a
combinatorial peptide library,
including but not restricted to a library made by phage display. Preferred
peptides in this aspect
of the invention are derived from proteins that are involved in natural inter-
cellular adhesion
and/or signal transduction processes, where it is more preferred that said
natural processes
involve at least one cell type of the hematopoietic system.
t o A typical nonlimiting example of a compound according to this aspect of
the invention is
a tissue culture plate coated with a mixture of antibodies directed against
molecules on the
surface of the virus-producing cell (e.g., retroviral envelope molecules) and
molecules on the
surface of the hematopoietic cell (e.g., the CD34 molecule present on the
membrane of primitive
hematopoietic cells}, or with bispecific antibodies directed against both cell
populations, or with
a mixture of synthetic peptides directed against both cell populations
(including, e.g., peptides
derived from cytokines known to act on said hematopoietic cells by binding to
a specific
receptor molecule).
The following are examples of direct methods which are used for bringing
hematopoietic
primate cells into close physical contact with recombinant retroviral vector
particles. These
2o embodiments of the invention make use of cell-free recombinant retroviral
vector preparations
derived from the culture medium of virus-producing cells that is harvested
according to standard
procedures known in the art. These procedures may include purification,
concentration, and the
like.
vi). Sedimentation of recombinant retrovirus vectors onto hematopoietic cells
of a
primate at increased gravitational force. Said increased gravitational force
is achieved by
incubating said hematopoietic cells in recombinant retroviral vector
containing medium while
spinning the container with the culture around an axis of rotation according
to the procedure
described in embodiment (ii). Said gravitational force should at least be
higher than the
minimal force needed to overcome the random diffusion of the recombinant
retroviral vector
3o and should not exceed the maximal gravitational force that allows
functional survival of the
recombinant retroviral vector and the hematopoietic cells from a primate,
where it is preferred
that the gravitational force is maximized. Usually, said gravitational force
will not exceed 2500
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g. the centrifugation time depends an the centrifugation speed and on the
height of the column
of culture medium above th.e hematopoietic cells, but typically does not
exceed two recombinant
retroviral vector half lives. Optionally, the procedure may be repeated
several times with fresh
recombinant retroviral vector containing medium.
vii). Electrodiffu,sion of recombinant retroviral vectors towards
hematopoietic cells of
a primate. By performing the cultivation of said hematopoietic cells of a
primate in recombinant
retroviral vector containing medium. in am electrophoresis unit said
hematopoietic cells of a
primate and recombinant re;trovirai vector particles that are both negatively
charged are forced to
move in the same direction towards the positive electrode and thus are
concentrated.
to Electrophoresis units useful for this aspect of the invention are known in
the art. Said
electrophoresis unit preferably cont;~ins two chambers separated by a semi-
permeable
membrane, with pore sizes that do not permit passage of said hematopoietic
cells of a primate
and said recombinant retaroviral vectors. In such a two-chamber
electrophoresis unit the
cultivation is performed in the chamber containing the negative electrode. The
voltage applied
t 5 between the electrodes is maximized, but kept below a value that causes
significant damage to
said hematopoietic cells of a primate and said recombinant retroviral vector
particles. To further
reduce said damage, said voltage may be applied periodically. Also in this
embodiment of the
invention, the procedure is typically not performed for longer than two
recombinant retroviral
vector half lives and may be repeated several times with fresh recombinant
retroviral vector
20 containing medium.
viii). Forcing recombinant retroviral vector particles towards hematopoietic
cells of a
erimate b~fluid flow. Said fluid flow is brought about by forcing the culture
medium
containing the recombinant retroviral vectors through a porous solid material
with pores large
enough in size to allow pau~sage of ;said culture medium but small enough in
size to prevent
25 passage of said hematopoietic cells of a primate. Said pores may or may not
allow passage of
the recombinant retroviral vectors. The force driving said fluid flow is
exercised as exemplified
above under iv). The rate of the fluid flow may range from the value that
compensates for
random diffusion of the recombinant retroviral vector to the maximal rate that
allows functional
survival of the recombinant retroviral vector particles and the hematopoietic
cells from a
3o primate. The time during which the fluid flow is maintained typically does
not exceed two
recombinant retroviral vecaor half lives. Optionally, the procedure may be
repeated severs!
times with fresh recombinant retroviral vector containing culture medium.
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26
ix). Culture of hematopoietic cells of a primate in recombinant retroviral
vector
containing medium in the presence of a compound that binds both said
hematopoietic cells of a
primate and said recombinant retroviral vector. Said compound has at least one
binding site for
said hematopoietic cell of a primate and at least one binding site for said
recombinant retroviral
s vector. The nature of said binding sites may be different or the same. Said
compound is
selected from or derived from the same molecules and materials characterized
above under (v).
Also in this embodiment of the invention it is preferred that said binding
site for a hematopoietic
cell of a primate has a binding preference for hematopoietic stem cells over
other types of
hematopoietic cells.
1o Also in this aspect of the invention, preferred compounds are derived from
or are
components of the natural hematopoietic microenvironment present in the bone
marrow of
animals. Further preferred compounds in this aspect of the invention comprise
parts that are
derived from antibodies or from peptides with a defined binding capacity as
characterized above
under (v). A typical nonlimiting example of a compound according to this
aspect of the
15 invention is a tissue culture plate coated with a mixture of antibodies
directed against the
envelope molecule on the surface of the recombinant retroviral vector and
molecules on the
surface of the hematopoietic cell, or with bispecific antibodies directed
against said vector and
cell, or with a mixture of synthetic peptides directed against said vector
(e.g., peptides derived
from the receptor for the retrovirus envelope molecule) and said cell.
2o x). Binding the recombinant retroviral vector to a compound that is
immobilized on
a solid suvnort material that is brousllt in close contact with the
hematopoietic cells of a primate
by anyone of the means exemplified in embodiments vi-viii or similar
procedures. Compounds
useful in this aspect of the invention have at least one binding site for said
recombinant
retroviral vector while being immobilized to said solid support material by
any physical or
25 chemical means. Said solid support materials include but are not restricted
to plastics, silicates,
metals, and the like. Additional examples of solid support materials include
agarose, sephrose,
sephadex, cellulose (acetate), DEAF-cellulose, polyacrylamide, polystyrene,
Tosylactivated
polystyrene, glass, gelatin, dextran, polyethylene, polyurethane, polyester. A
nonlimiting
example of this embodiment of the invention is the use of a plastic tissue
culture dish (the solid
3o support mhterial) coated by standard procedures known in the art with
antibodies directed
against the retrovirus envelope protein (the compound). Additional examples of
solid support
materials, in terms of physical structure: single or mufti-layer tissue
culture dish or flask, semi-
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27
permeable membrane, porous or non-porous beads including immunomagnetic beads,
and
(hollow) semi-permeable or non-pevrmeable fibers. Methods of coating and
coupling the
compound to the solid support materials include the following.
For polystyrene a siimple adsorption procedure can be followed. Protein
dissolved in
PBS is incubated for several hours ~~t room temperature with the solid support
material.
Subsequently, the coated s~~lid support material is washed once or several
times in PBS or in
PBS with 0.1% (w/v) irrelevant protein such as, e.g., albumin.
For other materials, covalent binding is preferred for efficient coating. Many
coupling
procedures and useful materials are known in the art and are commercially
available, e.g.,
1 o CNBr-activated sepharose (manufactured by Pharmacia) or agarose or dextran
can be used to
couple ligands containing amino groups by incubating them with ligand
dissolved in a
bicarbonate or borate buffi~r at high pH (preferably in the range of 8-10)
with a high salt content
(preferably approximately O.SM NaCI) for 2 hours at room temperature or for 10-
16 hours at
4°C. Subsequently, excess ligand i.s washed away with coupling buffer,
any remaining active
t 5 groups are blocked with, e~.g., 0.1 M Tris-HC 1 buffer pH 8.0 for 2 hours
at room temperature or
for 10-16 hours at 4°C, and sonically bound free ligand is washed away
by alternatively washing
with high and low pH buffer solutions such as, e.g., Tris-HC 1 buffer pH 8.0
with O.SM NaC 1
and O.1M acetate buffer p',H 4.0 with O.SM NaCl. Another example is
polystyrene activated by
p-toluenesulfonyl chloride; treatment (such as the Tosyiactivated Dynabeads M-
450
2o manufactured by Dynal). Any protein or glycoprotein can be chemically
coupled to this material
by incubating the solid sppport material with the ligand dissolved in a high
pH buffer such as
O.SM borate buffer pH 9.'.> for 24 hours at room temperature. Unbound ligand
is removed by
several washes with PBS with 0.1~% irrelevant protein (such as albumin). Many
alternative
coupling procedures and commercially available activated soluble support
materials useful in
25 the invention are known in the art see, e.g., Amity Chromatograph. A
Practical Approach,
1985 eds. P.D.G. Dean, VJ.S. Johnson, and F.A. Middle, IRL Press, Oxford,
pp.215). Apart
from a direct coupling of the ligand to the solid support material, the bond
can also be made via
a spacer molecule. Many reagents that can be used as spacer molecules have
been described.
Examples include bis-oxiranes, water soluble carbodiimides, SPDP, and
glutaraldehyde.
30 Finally, natural intermolecular interactions can be exploited to couple
proteins to a solid
support material, e.g., peptides containing a histidine-tag efficiently
interact with materials
containing nickel ions.
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The recombinant retroviral vector is bound to the compound by incubating a
preparation
of said recombinant retroviral vector in said tissue culture dish and said
hematopoietic cells of a
primate are brought in close contact with said recombinant retrovirus by
seeding said
hematopoietic cells of a primate in said tissue culture dish, where said
contact may be fiuther
s enhanced by, e.g., increasing the gravitational force.
Following the transfer procedures, it is not possible to determine the gene
transfer into
true hematopoietic stem cells in vitro, simply because there is no assay for
these cells.
However, more mature progenitor cells can be tested in standard colony assays
(e.g., McNiece et
al., Blood (1988) 72:191-195; Sutherland et al., Blood (1989) 74:1563-1570;
Breems et al.,
1o Leukemia (1994) 8:1095-1 I04). Furthermore, a candidate stem cell
population can be analyzed
phenotypically (Knaan-Shanzer et al., Gene Therapy (1996) 3:323-333). There
are several ways
of testing for gene transfer into these cells. When gene X encodes a
selectable marker gene,
clonogenia assays can be performed in the presence of a selective compound and
resistant
colonies can be scored to determine expression of the marker gene. If gene X
encodes a
t 5 molecule that can be stained with a fluorescent labeled antibody, or when
the product of gene X
converts a substrate into a fluorescent product, immunofluorescence or FACS
analysis can be
performed to demonstrate expression of the transgene. When gene X encodes a
transport
molecule that pumps a fluorescent substrate in or out of cells, expression of
gene X can be
measured by FACS analysis. Alternatively, isolated progenitor cell derived
colonies or cells
2o sorted on a FACS on the basis of their phenotype can be subjected to PCR
analysis spet;ific for
the introduced retroviral vector. The latter can be done on any vector
irrespective of the nature
of gene X.
Several of the embodiments i-x exemplified above may be combined to further
optimize
the transfer of gene X into the hematopoietic cells of a primate. It is,
therefore, to be understood
2s that any combination of said embodiments is also part of the invention. All
modifications
within the scope of the invention that may be contemplated by the skilled
artisan are also
claimed to be part of the present invention. All embodiments of the method of
the invention can
further be used after the hematopoietic cells of a primate have been enriched
for hematopoietic
stem cells, which is to be preferred in some cases. Enrichment of
hematopoietic cells of
3o primates for hematopoietic stem cells can be accomplished by various
methods known in the art.
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29
Below the invention is illustrated with practical examples. It is to be
understood that
only certain embodiments of the invention are illustrated and that the
examples should not be
considered restrictive in character.
EXAMPLES
Example (a) describes the production of virus-producing cells and recombinant
retroviral
vectors useful in the invention. In example (b) cells and vectors of example
(a) are shown to be
useful for the introduction of a gene. X into hematopoietic cells of primates,
without affecting
the in vivo regenerating capacity of the graft. Example (cl) describes a
procedure for the
to enrichment of isolated hematopoietic cells from a primate for hematopoietic
stem cells. In
example (c2) the usefulness of the invention for the introduction of a gene X
into enriched
hernatopoietic stem cells of a primate without affecting the in vivo
regenerating capacity of the
graft is demonstrated. Example (d) shows efficient transduction of
hematopoietic stem cells of a
primate by sedimentation of recombinant retroviral vectors onto said
hematopoietic stem cells at
t 5 increased gravitational force. Example (e 1 ) shows the production of
peptides useful in the
invention as recombinant retroviral vector binding compounds, and example (e2)
describes how
these peptides are used for the transfer of gene X into hematopoietic cells of
a primate according
to the invention. Example (fl ) disc:loses a procedure for the establishment
of a human stroma
cell culture derived from the natural microenvironment present in the human
bone marrow, and
2o example (f2) describes the use of this stroma cell culture as a binding
compound for the transfer
of gene X into haemopoietic cells of a primate.
Example a
Production of selectable stable recombinant retrovirus-producinss cells
25 In this practical example, u;~e was made of the retroviral vector construct
pLgXL(OMo+PyF 101 ) (Van Beusc;hem et al., ( 1990) J. Exp. Med. 172:729),
wherein A
represents the human cDrfA gene coding for adenosine deaminase (ADA). Twenty
micrograms
of this construct were transfected to the ecotropic packaging cell line GP+E-
86 (Markowitz et
al., (1988 J. Virol. 62:1120), according to the method described by Chen and
Okayam (Chen
3o and Okayama ( 1987) Mol. Cell. Biol. 7:2745). Prior to the transfection,
the GP+E-86 cells had
been cultured in medium containing 15 pg/ml hypoxanthine, 250 p.g/ml xanthine
and 25 pg/ml
mycophenolic acid, so as 'to select for the preservation of the DNA sequences
responsible for the
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production of the viral proteins. Transfectants that produce a functional
human ADA enzyme
were isolated by means of a selective culture in medium with a combination of
4 ~.M
xylofuranosyl-adenine (Xyl-A) and 10 nM deoxycoformycin (dCF) (Van Beuschem et
al.,
( 1990) J. Exp. Med. 172:729).
5 Then, with the thus obtained cells a ping-pong culture as described by Kozak
and Kabat
(Kozak and Kabat ( 1990) J. Yirol. 64:3500) was initiated. To that end, 5 x
103 transfectants
were mixed with an equal amount of GP+envAml2 amphotropic packaging cells
(Markowitz et
al., ( 1988) Virology 167:400) and cultured together in a-modified DMEM
(Dulbecco's Modified
Eagle's Medium) with 10% FCS (Fetal Calf Serum) and 8 p,g/ml polybrene. The
amphotropic
1 o packaging cells were also selected prior to use, for the preservation of
the DNA sequences
coding for the viral proteins (in the medium as described for GP+E-86 cells,
with 200 p,g/ml
hygromycin B added thereto). The culture was expanded for two weeks, at which
time the
amphotrolfic virus-producing cells were recovered using the resistance of
these cells against
hygromycin B. Individual GP+envAml2 clones that express functional human ADA
and
15 produce the viral proteins, were obtained by culturing limited cell numbers
in medium
containing all the above-mentioned components in the amounts mentioned. In
ail, 12 of such
clones were isolated and tested.
DNA analysis demonstrated that the clones contained several copies of the
retroviral
vector. The titer of the virus supernatants produced by the 12 clones was
measured by exposing
2o murine fibroblasts to dilutions of these supernatants and subsequently
determining the dumber
of fibroblasts that had acquired resistance against Xyl-A/dCF as a result
thereof. The different
clones produced between 3 x 103 and 2 x 105 infective virus particles per
milliliter supernatant.
The best clones produced 100 x more virus than the best amphotropic
LgAL(~IVIo+pyF 1 O 1 )
virus-producing cell line to date, which had been obtained via a single
infection with ecotropic
25 virus.
In order to obtain some idea about the most promising clone with regard to the
use in
bone marrow gene therapy procedures, rhesus monkey bone marrow was
cocultivated for three
days with each of the 12 virus-producing cell lines. Subsequently, the
preservation of the
capacity of the bone marrow to form hematopoietic colonies in vitro and the
infection efficiency
3o regarding~the hematopoietic precursor cells, which are at the origin of
these colonies, were
determined. With some of the clones, infection efficiencies of up to 40-45%
Xyl-A/dCF
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31
resistant precursor cells could be achieved, while none of the clones showed a
clear toxicity
towards these bone marrow cells.
On the basis of all aforementioned criteria, a cell line was chosen, which was
called
POAM-P1. This cell line vvas used to demonstrate in the practical example
described under b
the usefulness of the thus obtained virus procedures for the genetic
modification of the blood-
forming organ of primates.
Two further constn~cts based on the pLgXL(~Mo+PyF 1 O 1 ) retroviral vector
and
including further improvements were used, wherein gene X is the gene encoding
human
glucocerebrosidase. These vectors were designated IG-GC-2 and IG-GC-4 and
their
to construction is described ire detail ire patent application W096/35798, the
contents of which are
included herein by reference. IG-CrC-2 contains the full length human
placental
glucocerebrosidase (hCiC) cDNA, v~rhereas IG-GC-4 has a 160 nt deletion in the
3' untranslated
region of the hGC cDNA. Recombinant recombinant retroviral vector-producing
cell lines were
generated using the PA317 cell line with amphotropic host range (Miller and
Buttimore, Mol.
15 Cell. Biol. 6( 1986)2895-2502) and using the PGl 3 cell line with GaLV host
range (Miller et al.,
J. Virol. 65( 1991 )2220-22:?4) as described in patent application W096/35798.
The cell lines
were designated PA2 (PA=~ 17 with IG-GC-2 construct), PA4 (PA317 with IG-GC-4
construct,
PG2 (PG13 with IG-GC-2 construct), and PG4 (PG13 with IG-GC-4 construct).
To harvest batches of recombinant retroviral vector supernatants, T180 tissue
culture
2o flasks were inoculated with 1 x 106 virus-producing cells in 25 ml DMDM
(Gibco BRIT)
supplemented with 10% heat-inactivated fetal bovine serum (FBS) and said cells
were allowed
to grow to 90-100% confluency in ~4-5 days at 37°C, 10% C02 in a 100%
humidified
atmosphere. Next, the temperature was changed from 37°C to 32°C
for a period of 24 hours
before the medium was replaced with 50 ml fresh culture medium. After an
additional culture
25 period of 48 hours the vines supernatant was harvested, filtered through a
0.45 mm pore size
filter, aliquoted and stored at -80°C'.. The absence of replication
competent retrovirus (RCR)
was tested using a S+/L- foci test after amplification on mus dunni cells.
The recombinant retroviral vector titer issuing from the virus-producing cell
lines was
established on Gaucher type II fibnoblasts (GM1260). GM01260 cells were seeded
at a density
30 of 105 cells per 35 mm well (in 6-well plates) in culture medium further
supplemented with
polybrene (4 pg/ml; Sigma). Twenty-four hours later these cells were infected
with 1 ml of
recombinant retroviral vector supernatant after which the cells were cultured
and expanded for
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32
14 days as above. Next, genomic DNA was isolated as described by Stewart et
al., (Cell
38(1984)627-637). After digestion with NheI and Southern analysis using a 0.65
kb 32p-labeled
NcoI-BgQ hGC fragment according to standard procedures, both an hGC endogenous
fragment
( 19 kb) and a proviral DNA fragment of either 3.4 (IG-GC2) or 3.2 kb (IG-GC4)
were visible.
Comparison of signal intensities between the proviral DNA fragment and the
endogenous DNA
fragment by ImageQuant volumetric analysis after phosphor screen
autoradiography using a
Molecular Dynamics PhosphorImager 400A revealed a ratio of 0.8 (PA2), 0.3
(PA4), 0.4 (PG2),
and 0.2 (PG4). Since the hybridization signal of the endogenous band
represents 2n DNA, on
average 1.6, 0.6, 0.8, and 0.4 provirus copies per cell were present,
respectively. Taking into
1 o account that the seeded GM01260 cells probably divided once before the
virus supernatant was
applied, approximate virus titers of 3 x 105, 1 x 105, 2 x 105, and 8 x 104
were calculated for
PA2, PA4, PG2, and PG4, respectively.
Example b
Preclinical test of a bone marrow ;gene therapy procedure in rhesus
monkeys with the cell line POAM-P 1 described in Example a, above
Rhesus monkey bone marrow was taken by puncturing the upper legs. The bone
marrow
so obtained was suspended in HBSS/Hepes with 100 units heparin and 100 ~,g/ml
DNase I.
Cells having a density lower than 1.064 g/ml were obtained by successively
performing a Ficoll
2o separation and a BSA-density gradient centrifugation (Dicke et al., (1969)
Transplantation
8:422). These operations resulted in an enrichment of the cell population for
hematopoietic
stem cells by a factor of 10-20. The thus obtained bone marrow cells were
introduced, in a
concentration of 106 cells per ml, into high glucose (4.5 g/Iiter) a-modified
DMEM, containing
S% heat-inactivated monkey serum, 15 mg/ml BSA (Bovine Serum Albumin), 1.25 x
10'5 M
Na2Se03, 0.6 mg/ml iron-saturated human transferrin, 1 ~,g/ml of each of the
following
nucleosides: adenosine, 2'-deoxyadenosine, guanosine, 2'-deoxyguanosine,
cytidine, 2'-
deoxycytidine, thymidine and uridine, 1.5 x 10-5 M linoleic acid, I .5 x 10'5
M cholesterol, 1 x
10-4 M ~i-mercaptoethanol, 0.4 ~tg/ml polybrene, 100 pg/ml streptomycin, 100
U/ml penicillin
and 50 ng/ml of the recombinant rhesus monkey hematopoietic growth factor IL-3
(Burger et
3o al., (1990 Blood 76:2229). The thus obtained cell suspension was seeded at
a concentration of
2 x 105 cells per cm2 onto a 70-80% confluent monocellular layer of POAM-P 1
cells, which had
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33
shortly before been exposed. to 20 Gray y-radiation. The bone marrow was
cocultivated with the
POAM-P1 cells for 90 h at :37°C in a moisture-saturated atmosphere of
10% C02 in air.
For the duration of the cocultivation, the rhesus monkey that had donated the
bone
marrow was conditioned four the autologous reception of the cocultivated bone
marrow by means
of total body irradiation with 10 Gray x-rays, divided over two equal
fractions at an interval of
24 h, performed, respectively, 2 days and 1 day prior to the transplantation.
On the day of the
transplantation, the cocultivated bone marrow was harvested from the culture,
including the
bone marrow cells that had adhered to the POAM-P 1 cells or cells that had
adhered to the
plastic of the culture bottle during cultivation. The latter cells were
obtained by means of a
1 o trypsinization. A monocellular cell suspension was prepared in a
physiological salt solution
with 10 pg/ml DNase I and infused unto a peripheral vein of the donor monkey.
In order to determine the in vivo regeneration capacity of the cocultivated
bone marrow,
use was mhde of the semi-qluantitative assay described by Gerritsen et al.,
(Gerritsen et al.,
(1988) Transplantation 45:301). Thus method is based on the observation that
the rate at which
15 circulating red and white blood cells regenerate after transplantation of
autologous bone marrow
cells in lethally irradiated rhesus monkeys depends on the size of the
transplant. In particular
the kinetics of the appearance of the precursors of red blood cells
(reticulocytes) is a good
standard in this connection.. By determining hematological values in the blood
system of the
monkeys at regular intervals after the transplantation, it could be
established (using the relation
2o described by Gerritsen) that the modified bone marrow had preserved
sufficient regenerative
capacity and the cocultivati.on therefore had no toxic side effects.
Analysis at the DN~~ level made it clear that long periods (up to more than a
year) after
the transplantation, the introduced provirus could be traced in various blood
cell types
(mononuclear cells and granulocytes). Especially the presence of the
introduced gene in the
25 granulocytes is considered of great importance. Since granulocytes, after
being generated in the
bone marrow, remain in the blood stream only a few hours before being broken
down, the
presence of the human AD.A in these cells demonstrates that a year after
transplantation the bone
marrow still contains very ;primitive; cells that give rise to the formation
of ripe blood cells.
Also, functional expression of the uztroduced human ADA gene in ripe blood
cells could be
3o demonstrated. These results constitute clear proof of the fact that through
the invention
described here stable genetic modification of the hematopoietic system of
primates can be
obtained.
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34
Example c
Preclinical test of a bone marrow gene therapy procedure in rhesus
monkeys which utilizes purified hematopoietic stem cells
c 1 ) Enrichment of primate bone marrow CD34+CD 11 b-stem cells
Rhesus monkey bone marrow having a density lower than 1.064 g/ml was
obtained as described above under Example b. This cell population was
successively depleted
for cells carrying the monocytes/granulocytes-marker CD 11 b and enriched for
cells carrying the
stem celI/precursor cell-marker CD34. This was performed using immunomagnetic
beads,
1 o which had been made as follows: first, tosyl-activated polystyrene
magnetic beads (Dynabeads
M-450; Dynal, Oslo) were incubated for 24 h in a 0.5 M borate solution pH 9.5
with 1.25 pg
protein A (Pharmacia, Uppsala) per 106 beads. After frequent washing in PBS
containing 0.1
BSA, to the beads, now protein A-coupled, saturating concentrations of
monoclonal antibodies
(anti-CD1 lb: Mol, Coulter Clone, Hialeah, Fl; anti-CD34: ICH3, 43) were bound
by incubating
15 for 30 min at room temperature. Finally, the beads were frequently washed
in HBSS/Hepes and
stored at 4°C until use. The bone marrow cells were incubated for 20
min at 4°C with 7 anti-
CD 11 b beads per cell in a concentration of 5 x 10' cells/ml at a maximum.
Unbound CD 11 b-
negative cells were stripped from beads and CD 11 b-positive cells bound
thereto, using a
magnetic particle collector (MPC; Dynal) and washed in HBSS/Hepes. The thus
obtained cells
2o were incubated for 20 min at 4°C with 5 anti-CD34 beads per cell
again in a concentration of S
x 10' cells/ml at a maximum. After removal of the CD34-negative cells using
the MPC, the
bound CD34-positive cells were recovered by means of a competitive elution
with an excess of
immunoglobulins. To that end, the beads with CD34-positive cells were
incubated for 1 h at
37°C in HBSS/Hepes with 25% bovine plasma (Gibco, Paisley) and 500 U/mi
heparin.
c21 Introduction of the construct pL~~XL(OMo+pyF101) described under a into
rhesus monkey CD34+CD 11 b- stem cells
The introduction of the human ADA gene into rhesus monkey CD34+CD 11 b-
stem cells and the autologous transplantation procedure were performed as
described under
3o Example b above, the only difference being that the cocultivation was
performed with the
previously described cell line POC-1 (Van Beuschem et al., (1990) J. Exp. Med
172:729). As
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noted, this cell line is unstable and not very suitable for large-scale use.
For this present
experiment, use could still be made of an early passage which does not have a
reduced titer.
After transplantation all blood cell types regenerated completely, which
demonstrates
that the gene transfer procedure can also be performed on CD34+CD11- stem
cells without
s toxic side effects. The presence of the provirus in mononuclear blood cells
and in granulocytes
could also be demonstrated in these monkeys during the entire experimental
period (at this point
266 days and 280 days after transplantation in two monkeys) which is still in
progress.
Expression of the function2il human ADA enzyme could also be demonstrated in
blood cells of
these monkeys. The enrichment for hematopoietic stem cells prior to the gene
transfer did not
t o have any demonstrable effect on the efficiency of the gene transfer to
stem cells. This
experiment therefore demonstrates that the results as described under b) can
also be achieved
when the bone marrow has been stripped from most riper cell types, which is
preferred in some
uses of genetic modification of bone; marrow cells.
t 5 Example d
Introduction ~nf the IG-GC constructs described under !a) into human
CD34+ hernato oietic stem cells by increased ,~xavitational force
Bone marrow cells were obtained by aspiration of the iliac crest of normal
healthy
donors or of a patient with Non-Hodgkin Lymphoma. Mononuclear cells were
obtained by
2o Ficoll gradient separation according to standard procedures. CD34+
hematopoietic step cells
were isolated using a magnetic antibody separation system (Mini Macs, Milteny)
according to
the procedures supplied by the manufacturer. This procedure yielded 60-95%
pure CD34+
populations with recoveries ranging from 50-90% of the CD34+ cells present in
the total bone
marrow aspirate.
z5 Recombinant retroviral vector supernatant of the virus-producing cell lines
PA2, PA4,
PG2 and PG4 obtained as described under Example (a) was used for the
transduction of the
isolated human CD34+ cells. Said iisolated CD34+ cells were seeded at a cell
density of 1 x 105
cells/cm2 in 24-well tissue culture plates (Greiner) in 400 pl virus
supernatant supplemented
with 50 ng/ml interleukin-=1 (Sandoz) and 4 ~g/ml protamine sulfate (Novo
Nordisk Pharma).
3o The plates:~were subsequently centrifuged for 2.5 hours at 1100 g at room
temperature, either
once or four times (once daily after refreshing the virus supernatant). After
each centrifugation,
the cultures were placed overnight at 37°C, 10% C02 in a 100%
humidified atmosphere. In a
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36
control experiment, the cells were cultured for four days as above without the
2.5 hours
centrifugation steps. Instead, the recombinant retroviral vector medium was
refreshed daily
after a 5 minute centrifugation of the cultures at 200 g. The theoretical
multiplicity of infection
of functional recombinant retroviral vector particles in the total culture
medium over
hematopoietic target cells at the start of the procedure after each
supernatant addition was 1.2,
0.4, 0.8, and 0.3 for PA2, PA4, PG2 and PG4 virus, respectively. As a control
virus preparation,
culture supernatant of the IGvp010 cell line (see patent application
W06/35798) was used that
contains pLgXL(~Mo+pyF 1 O 1 ) derived recombinant retrovirus vectors carrying
the human
multi-drug resistance (MDRI ) gene at a titer of approximately 105 particles
per ml as established
1 o by vincristine resistant colony formation of human bone marrow cells.
In experiment 1, CD34+ cells from bone marrow of a non-Hodgkin lymphoma
patient
were transduced by four incubations with PA2, PG4, or IGvp010 virus
supernatant with or
without transduction enhancement by centrifugation. After the transduction
procedure,
transduced CD34+ cells were seeded in 1 ml DMEM (Gibco BRL) with 10% FBS
supplemented
t5 with 200 ng/ml SCF, 100 ng/ml IL-6, 100 ng/ml II,-3, 100 U/ml GM-CSF, and
100 ng/ml G-
CSF. After a 10 day culture period at 37°C, 10% C02 in a 100%
humidified atmosphere the
expanded cells, representing the mature myeloid progeny of the transduced
CD34+ cell
population, were washed once with PBS and lysed in buffer containing 50 mM
potassium
phosphate buffer, pH 6.5, 0.1% Triton X-100. Following sonication and
centrifugation at 4°C,
2o the clear supernatant was transferred to new tubes, protein concentrations
were measured (DC-
Biorad kit) and lysates were stored at -20°C.
Glucocerebrosidase activity was determined with either 4MU-b-glucoside (Sigma)
or
PNP-b- glucoside (Sigma) as artificial substrate on 20 mg total protein of
transduced cells
according to described procedures (Aerts et al., Eur. J. Biochem. 150(1985)565-
574; Havenga
25 et al., BioTechniques 21 ( 1996) 1004-1007).
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37
TABLE 1
Comparison of 4z supernatant transducNon procedure
to ~~ a centrifugation enhanced transduction
Recombinant Retrovirus
Vector Supernatant Relative hGC Activity PCR-positive CFU-GM
~iupernatant Centrifu ag ton Supernatant Centrifugation
IGvp010 (negative ( 1 ) ( 1 ) 0/24 (0%) 0/24 (0%)
control)
PA2 hGC vector 1 1 1/24 (4%) 5/24 (21%)
PG4 hGC vector 1.3 4.5 3/24 (13%) 3/24 (13%)
Table 1 shows the relative glucocerebrosidase activity data of this
experiment, where the
results of cells subjected to ~transduction with the IGvp010 retrovirus vector
were set at a value
of 1. As can be seen, an increase in hlGC activity could not be detected
following transduction
with PA2 virus, with or without centrifugation. 1n contrast, PG4 virus
transduction could be
measured by functional hG(: activity which was 3.5-fold increased following
centrifugation (4.5
1 o versus 1.3 in the control). Successful transduction was further confirmed
by performing PCR
specific for the IG-GC-2 and IG-GC-4 constructs on CFU-GM clonogenic
progenitor cell
derived colonies. CFU-GM: were obtained by seeding 5000 transduced CD34+ cells
in 1 mI of
methylcellulose medium with cytokines (Methocult GF H4534; Stemcell
Technologies, Inc.,
Vancouver, Canada) in 6-well plates. After 14 days, individual colonies were
picked anti DNA
was isolated as described (van Beusechem et al., Proc. Natl. Acad Sci. USA
89(1992)~1640-
7644). PCR analysis was performed on this suspension using oligonucleotide
primers 5'-
CAGCCCATGTTCTACC~~C-3' and 5'-GGATCCCTAGGCTTTTGC-3'. A SO ~1 PCR reaction
typically contained 25 pmol of each oligonucleotide, 3% DMSO, 5 pl 10-times
concentrated
buffer provided with the erezyme, 20 pmol dNTP, and 0.25 Units SuperTaq
(Promega). The
2o cycler program consisted of S min. 95°C predenaturation followed by
26 cycles of each 45 sec.
95°C, 1 min. 54°C, 1 min. ~~2°C. The program was ended by
an elongation step of 10 min. at
72°C. Of the PCR product, 10 pl was run on a 1.5% agarose gel,
transferred to a membrane and
hybridized with a 0.4 kb 32p-labeled BamHI hGC fragment according to standard
procedures.
As can be seen in Table 1, al transductions with hGC retrovirus vectors
yielded PCR-positive
colonies, whereas transductions with the MDR1 control vector did not.
In experiment 2, PA2, PA4, and PG2 recombinant retroviral vector supernatants
(and
IGvp010 control supernatant) were used to transduce CD34+ cells from normal
healthy donor
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38
bone marrow. The centrifugation procedure was performed either once or four
times on
subsequent days as above. One day after the transduction procedure, CFU-GM
were plated for
PCR analysis as above. As can be seen in Table 2, all transductions led to
high percentages of
hGC-vector containing CFU-GM even after a single 2.5 hour centrifugation step.
TABLE 2
Comparison of a single centrifugation enhanced transduction procedure to
four repeated centrifugation enhanced transduction procedures
Recombinant Retrovirus
Vector Supernatant PCRpositive CFU-GM/number tested (5)
Single transduction Four rounds of transduction
IGvp010 control vector 0/20 (0%) 0/20 (0%)
PA2 hGC vector 6/20 (30%) 5/20 (25%)
PA4 hGC vector 5/20 (25%) 6/20 (30%)
.PG2 hGC vector 9/20 (45%) 10/20 (50%)
1 o Example a
E~cient transfer of gene X into human CD34+ hematopoietic stem cells
urine recombinant retroviral vectors bound to a tissue culture dish
el ) Production of peptides derived from receptors for retroviruses (GLVR)
The Gibbon ape Leukemia Virus Receptor (GLVR) proteins are transmembrane
15 molecules expressed on the surface of mammalian cells. Their primary
function is imp~Ort of
inorganic phosphate and sodium. To date, two different but homologous
receptors have been
described by means of expression cloning of complementary DNA copies of their
marine and
human mRNA counterparts (Johann, et al., J. Virol. 66( 1992) 163 S-1 b40; van
Zeij I, et al.,
Proc. Natl. Acad Sci. USA 91(1994)1168-1172; Weirs and Tailor, Cell
82(1995)531-533). The
2o cDNA predicted amino acid sequences and deduced hydropathy plots suggest
that both these
GLVR1 and GLVR2 proteins traverse the cellular membrane 10 times and have 5
extracellular
loops and 4 intracellular loops. The human GLVRI receptor confers permissivity
to Gibbon ape
Leukemia Virus and Feline Leukemia Virus-B, whereas the human GLVR2 or
amphotropic
virus receptor confers permissivity to amphotropic Marine Leukemia Viruses
carrying the
25 4070A or ~IOAI envelope molecules. The GLVR1 homologues from different
rodent species
have small amino acid differences in their 4a' extracellular domain which
determine virus
susceptibility of a cell. Recombinant chimeras between GLVR1 and GLVR2
proteins suggest
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39
that the 4~' extracellular domain in (iLVRl is involved in virus binding and
infection.
Therefore, we have synthesized peptides encompassing sequences from the 4'h
extracellular
domain of GLVR1 and GL'VR2 using Fmoc chemistry (performed under contract at
Research
Genetics, Inc. Huntsville, A,L, USA;I. The amino acid sequences of said
peptides read from N-
terminus to C-terminus: LVYDTGDVSSKV and LIYKQGGVTQEA for GLVRI and GLVR2,
respectively.
For certain applications of tl:'e invention, the C-terminus of said peptides
is extended
with 6 Histidine-residues. 'This enables coupling to solid support materials
via pickle
molecules.
to
e21 The use of (iLVR-peptides as recombinantretrovirusvector binding compounds
to enhance the transfer of gene X into human hematopoietic stem cells
Non-tissue culture dishes, i.e. culture dishes not treated to enhance cell
adherence
(35 mm; Greiner) are incubated for 2 hours at room temperature with 2 ml 100
NM GLVR1 or
15 GLVR2 peptide in phosphate buffered saline (PBS). This solution was
prepared from a 10 mM
stock in DMSO. Next, the dishes are washed once with PBS. Two ml recombinant
retrovirus
supernatant harvested from the PA2 cell line and from the PG4 cell line as
described under (d) is
incubated at 4°C for 2 how's on GL'VR2 or GLVR1 peptide coated dishes,
respectively. This
procedure is repeated twice;. Optionally, the thus coated dishes are washed
with PBS with 1%
20 (w/v) hwnan serum albumin (PBS/IiAS) and stored at -80°C.
Hwnan CD34+ henaatopoietic stem cells are obtained as described under Example
(d)
above, are suspended 1 x 106 cellshnl in nViDM (Gibco BRL) supplemented with
50 ng/ml
interleukin-3 (Sandoz), 5°/. heat-inactivated autologous human serum, 4
pg/ml protamine sulfate
(Novo Nordisk Pharma) and 100 U/ml penicillin (Gist-Brocades) and are cultured
for 48 hours
25 at 37°C, 10% C02 in a lOd~% hwnidified atmosphere in non-tissue
culture dishes. Next, the
cultured cells are placed in the GLVR peptide and recombinant retroviral
vector coated dishes in
their original culture medium (2 ml/dish) and cultured for another 24 hours at
37°C, 10% C02 in
a 100% humidified atmosphere. Aver this culture, ail cells including any
adherent cells are
harvested, washed once in PBS/HA,S, and used for analysis of gene transfer or
for
3o transplantaltion by infusion into a peripheral vein.
Our invention shoves in an example that bone marrow cells cocultivated with
the virus-
producing cells described here are capable of genetically modifying the
hematopoietic system of
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primates after autologous transplantation. This modification was observed for
a prolonged
period in several blood cell types including granulocytes, which have a very
short life time
(approximately 8 hours). With the method described by us, these results can
also be obtained
when the bone marrow has previously been enriched for hematopoietic stem cells
by removal of
most other (riper) bone marrow cells. These data demonstrate our capacity to
infect very
primitive cells and show that it is possible to carry out gene therapy using
such modified bone
marrow cells.
Example of
Enhanced transfer of gene X into human CD34+ ~emopoietic stem cells using
t o recombinant retrovirus vectors in the~resence of human bone marrow stroma.
fl ) Establishment of human bone marrow stroma
Bone marrow mononuclear cells from healthy donors are obtained as described in
example (d). Five x10' cells are seeded in T75 Nunclon culture flasks (Nunc,
Roskilde,
t s Denmark) in 10 ml DMEM (Gibco) supplemented with 10% heat-inactivated FCS,
and cultured
at 37°C, 10% C02 in a 100% humidified atmosphere. Twenty-four hours
later the entire
medium, including all non-adherent cells, is removed and replaced with the
same medium
further supplemented with 2 mM L-glutamine (Gibco), 10~ M (3-mercaptoethanol
{Merck,
Darmstadt, Germany), and 10-5 M hydrocortisone (Sigma)("stroma medium"). Once
a week, the
2o stroma medium is replaced with fresh stroma medium. After 3-S weeks, a
confluent ~onolayer
of cells is formed. Thereafter, confluent monolayers are trypsinized with
Trypsin-EDTA
solution (Gibco) and split 1:10 in stroma medium each time after reaching
confluence. Each
reseeding step is regarded as one passage and includes 3-4 cell doublings.
Three individual
stroma lines were established and these have now been cultured for 40, 40, and
65 passages,
25 respectively. The three lines exhibited similar functional properties in
supporting maintenance
of human haemopoietic stem cells throughout the entire study, i.e., at least
during the culture
period ranging from passage 5 to passage 30.
f2) The use of human bone marrow stroma as a bindin~compound to enhance the
retroviral vector-mediated transfer of gene X into human haemopoietic stem
cells.
3o Twenty-four-well tissue culture plates are precoated with 0.3% gelatine
(Sigma) in PBS
for 16 hours at 4°C. Stroma cells are seeded 2 x 105 cells/well into
these plates. After 1-3 days,
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41
confluent stroma monolayers are irradiated with 25 Gy Y-radiation. Immediately
thereafter, the
irradiated stroma monolayers are used to support retroviral vector-mediated
gene transfer.
Serum-free recombinant retrwirus supernatant is harvested from the cell line
IGvp010
(see Patent Application W096/35798) that produces pLgXL(OMo~PyF101) derived
recombinant retroviral vectors carrying the MDR1 gene, either in IGTM (alpha-
modified
DMEM containing 1.5% B;~A (Sigma), 1 ~gml of each of the nucleosides
adenosine, 2'-
deoxyadenosine, guanosine, 2'-deoxyguanosine, cytidine, 2'-deoxycytidine,
thymidine, and
uridine (ail Sigma), 1.5 x 10'' M Na2Se03 (Sigma), 0.6 mg/ml iron-saturated
transferrin
(Behring, Marburg, Germairy), 1.5 x '°'' M linoleic acid (Sigma), 1 x
10~ M 2-mercaptoethanol
to (Merck, Darmstadt, Germa~ry) with 5% fetal calf serum (FCS) or in serum-
free StemPro-34
SFM Complete Medium (Gibco RB:L Life Technologies, Grand Island, N~. The
retroviral
vector supernatant is flash-fiozen and stored at -80°C until use.
Human CD34+ bone marrov~r cells are obtained as described in example (d). They
are
suspended 1 x 106 cells/ml in IGvp010 supernatant in IGTM with 5% FCS or
StemPro-34 SFM
15 Complete Medium supplemented with SO ng/ml interleukin-3 (Gist-Brocades,
Delft, The
Netherlands) and 1.6 pg/ml protamine HC1 {Kabi Pharmacia, Woerden, The
Netherlands) and
are seeded 5 x 105 cells per well onto the irradiated stroma monolayers.
Control cultures are
started with the same cell s»spensions in culture dishes without stroma
monolayers. The cells
are cultured for four days at 37°C, 10% C02 in a 100% humidified
atmosphere, and each day the
2o complete medium is replaced by fre;;h IGvp010 supernatant and supplements.
On day ~, all
cells are harvested by trypsinization as above, and are used for gene transfer
analysis or
transplantation.
For analysis of gene transfer into haemopoietic stem cell and progenitor cell
populations,
the cells are stained with a phycoerydlrin-conjugated anti-CD34 MoAb and with
a cocktail of
25 fluorescein isothiocyanate-c;onjugate~d Moabs directed against CD38, CD33,
and CD71 as
described (ICnaan-Shanzer Eat al., Gene Therapy (1996) 3:323-333). Populations
of
CD34b"~"CD33,38,71"'8°""e' cells,
CD34°°s"'~'CD33,38,71p°S"'°' cells,
CD34"'se"°eCD33,38,71~°S"~°' cells, and
CD34"'g""''CD33,38,71"'g°"°' cells are each sorted
separately on a FACStar Ph~s flow cytometer (Becton Dickinson, Mountain View,
CA).
3o Aliquots 6f the sorted samples are used for reanalysis to determine the
purity of the sorted cells.
The presence of the recombinant retroviral vector genome in the sorted cells
is determined by a
semi-quantitative PCR assay. To this end, untransduced bone marrow mononuclear
cells are
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42
added to the sorted cell samples to reach a total of 106 cells per sample.
'The cells are pelleted
by centrifugation and DNA is isolated from these cells as described (van
Beusechem et al.,
Proc. Natl. Acid Sci. USA ( 1992) 89:7640-7644). The isolated DNA
concentration is measured
using PicoGreen DNA Quantitation reagent (Molecular Probes, Eugene, OR) and of
each isolate
five independent titrations are prepared containing DNA equivalents down to 10
cells per
sample. All samples are subjected to PCR analysis specific for the human MDRI
cDNA gene.
The sequences of the primers used are: 5'-GTCACCATGGATGAGATTGAG-3' (upstream
primer) and 5'-CCACGGACACTCCTACGAG-3' (downstream primer). The reaction
conditions are: 10 mM Tris-HC 1 pH 9.0, S 0 mM KC l, 0.01 % (w/v) gelatin, 0.1
% Triton X-100,
1.5 mM MgCl2 with 200 N.M of all four dNTPs, 200 pM of both primers, and 0.25U
SuperTaq
polymerise (HT Biotechnology Ltd. Cambridge, UK) in a total volume of 50 p,l.
Forty cycles of
1 minute at 94°C, 1 minute at 55°C and 1 minute at 72°C
are performed in 96-well plates using
a Biometra UNO-Thermoblock thermocycler. The reaction products are separated
on 0.8%
agarose gel, blotted, and subject to Southern analysis with human MDR1 gene
specific probes
i 5 according to standard procedures (Sambrook, Fritsch, and Maniatis ( 1989)
Molecular Cloning.
A Laboratory Manual. Second edition, Cold Spring Harbor Laboratory Press, Cold
Spring
Harbor, N~. The frequency of PCR-positive cells is determined on the basis of
the detection of
specific amplification products in the five independent titrations. The
outcome of this analysis
is corrected for a possible contribution to the PCR signal by any
contaminating cells with a
2o different phenotype using the data from the FACS reanalysis of the sorted
samples. (See Knaan-
Shanzer et al. Gene Therapy (1996) 3:323-333.
All publications and patent applications mentioned in this specification are
indicative of
the level of skill of those skilled in the art to which this invention
pertains. All publications and
patent applications are herein incorporated by reference to the same extent as
if each individual
2s publication or patent application was specifically and individually
indicated to be incorporated
by reference.
The invention now having been fully described, it will be apparent to one of
ordinary
skill in the art that many changes and modifications can be made thereto
without departing from
the spirit or scope of the appended claims.
