U.S. patent application number 10/164076 was filed with the patent office on 2003-09-04 for methods and compositions for genetically modifying primate bone marrow cells.
Invention is credited to Valerio, Domenico, Van Beusechem, Victor Willem.
Application Number | 20030166285 10/164076 |
Document ID | / |
Family ID | 19859782 |
Filed Date | 2003-09-04 |
United States Patent
Application |
20030166285 |
Kind Code |
A1 |
Valerio, Domenico ; et
al. |
September 4, 2003 |
Methods and compositions for genetically modifying primate bone
marrow cells
Abstract
A method is provided for genetically modifying primate bone
marrow cells, comprising isolating bone marrow cells from a primate
and, by means which enhance the local concentration of retroviral
particles, contacting the isolated bone marrow cells to cells that
produce a recombinant amphotropic retrovirus with a genome based on
a retroviral vector that contains the genetic information to be
introduced into the bone marrow cells. Recombinant amphotropic
retrovirus-producing cells, suitable for use in this method also
are provided, as are genetically modified primate bone marrow cells
with the capacity for regeneration in vivo.
Inventors: |
Valerio, Domenico; (Leiden,
NL) ; Van Beusechem, Victor Willem; (Amsterdam,
NL) |
Correspondence
Address: |
TRASK BRITT
P.O. BOX 2550
SALT LAKE CITY
UT
84110
US
|
Family ID: |
19859782 |
Appl. No.: |
10/164076 |
Filed: |
June 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10164076 |
Jun 4, 2002 |
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08820479 |
Mar 18, 1997 |
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6472212 |
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08820479 |
Mar 18, 1997 |
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08211342 |
Jun 21, 1994 |
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5612206 |
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08211342 |
Jun 21, 1994 |
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PCT/NL92/00177 |
Oct 5, 1992 |
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Current U.S.
Class: |
435/456 ;
435/372 |
Current CPC
Class: |
C12N 2740/13022
20130101; C12N 2740/13045 20130101; C12N 2810/6054 20130101; C07K
14/005 20130101; C12N 2740/13043 20130101; C12N 2810/855 20130101;
C12N 9/78 20130101; C12N 15/86 20130101; A61K 48/00 20130101; A61P
7/00 20180101 |
Class at
Publication: |
435/456 ;
435/372 |
International
Class: |
C12N 015/867; C12N
005/08 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 4, 1991 |
NL |
9101680 |
Claims
What is claimed is:
1. A method for genetically modifying primate hematopoietic stem
cells, said method comprising: combining isolated primate
hematopoietic stem cells which can undergo replication with a
recombinant retrovirus comprising a genome based on a retroviral
vector into which a gene X is inserted, wherein said combining uses
means whereby said hematopoietic stem cells and said recombinant
retrovirus are brought into close physical contact, so that chance
of infection of said hematopoietic stem cells by said recombinant
retrovirus is significantly enhanced.
2. The method according to claim 1, wherein said means comprises
co-cultivation of said hematopoietic stem cells with cells which
produce said recombinant retrovirus.
3. The method according to claim 1, wherein said means are physical
means.
4. The method according to claim 3, wherein said physical means
comprise centrifugation of medium containing said hematopoietic
stem cells and said recombinant retrovirus.
5. The method according to claim 1, wherein said means comprise use
of a compound which has binding affinity for both said recombinant
retrovirus and said hematopoietic stem cells.
6. The method according to claim 5, wherein said compound which has
binding affinity for'both hematopoietic stem cells and said
recombinant retrovirus is a component of an extracellular matrix
present in a natural hematopoietic environment.
7. The method according to claim 1, wherein said retroviral vector
is derived from a viral MuLV vector.
8. The method according to claim 7, wherein said retroviral vector
comprises as operably linked components with said gene X, a 5' LTR
and a modified 3' LTR, wherein said 5.about.LTR and said 3' LTR are
derived from a ,Jiral MuLV vector, and a 5' part of a MuLV gag
gene.
9. The method according to claim 8, wherein said MuLV vector is
derived from a Mo-MuL.Yen. vector and wherein said modified 3' LTR
is a hybrid LTR that contains a PyF101 enhancer instead of a
Mo-MuLV enhancer.
10. The method according to claim 9, wherein said retroviral vector
comprises vector pLgXL(AMo+PyFI01).
11. The method according to claim 2, wherein said cells which
produce said recombinant retrovirus are mammalian cells which
express MuLV gag, pol and env genes.
12. The method according to claim 11, wherein said env gene is
derived from an amphotropic MuLV.
13. The method according to claim 11, wherein said MuLV gag, poi
and env genes are contained in at least two different eukaryotic
expression vectors.
14. The method according to claim 11, wherein each of said
expression vectors is associated with a selectable marker gene.
15. The method according to claim 11, wherein said mammalian cells
are GP+envAM 12 cells.
16. The method according to claim 10 wherein said retrovirus is an
amphotropic retrovirus.
17. The method according to claim 16, wherein cells which produce
said recombinant amphotropic retrovirus contain several copies of
said retroviral vector.
18. The method according to claim 2, wherein said cocultivation is
in medium which contains serum and at least one hematopoietic
growth factor.
19. The method according to claim 2, wherein following said
co-cultivation, non-adherent bone marrow cells are harvested
together with adherent bone marrow cells.
20. A cell which produces a recombinant retrovirus comprising a
genome based on a retroviral vector into which is inserted a gene X
for introduction into isolated primate bone marrow cells by the
method of cocultivating isolated primate bone marrow cells with
said recombinant retrovirus.
21. The cell of claim 20, wherein said retroviral vector is derived
from a viral MuLV vector.
22. The cell of claim 21, wherein said retroviral vector comprises
as operably linked components with said gene X, a 5' LTR and a
modified 3' LTR, wherein said 5' LTR and said 3' LTR are derived
from a viral MuLV vector, and a 5' part of a MuLV gag gene.
23. The cell of claim 22, wherein said viral MuLV vector is derived
from a viral Mo-MuLV vector and said modified 3' LTR is a hybrid
LTR that contains a PyF101 enhancer instead of a Mo-MuLV
enhancer.
24. The cell of claim 23, wherein said retroviral vector is
pLgXL(&Mo+PyF101), wherein X represents said gene X.
25. The cell of claim 18, wherein said retrovims is an amphotropic
retrovirus.
26. The cell of claim 25, wherein said cell is a mammalian cell
which expresses MuLV gag, pol and env genes.
27. The cell of claim 26, wherein said env gene is derived from an
amphotropic MuLV.
28. The cell of claim 26, wherein said MuLV gag, poi and env genes
are contained in at least two different eukaryotic expression
vectors.
29. The cell of claim 26, wherein each of said expression vectors
are associated with a selectable marker gene.
30. The cell of claim 26, wherein said cells are GP+envAM]2
cells.
31. The cell of claim 26, wherein said recombinant retrovirus
contains several copies of said retroviral vector.
32. A process for producing genetically modified primate
hematopoietic cells with long term repopulating capacity, said
method comprising: collecting both adherent and nonadherent
genetically modified primate bone marrow cells from a culture in
which unmodified primate bone marrow cells were combined with a
recombinant retrovirus under conditions whereby retroviral particle
concentration in the immediate vicinity of said unmodified bone
marrow cells was enhanced so as to increase efficiency of
transduction of said bone marrow cells.
33. Genetically modified primate hematopoietic cells with long term
repopulating capacity comprising: a plurality of adherent and
nonadherent genetically modified primate bone marrow cells.
34. Genetically modified primate hematopoietic produced by the
method of claim 1.
35. A method for genetically modifying primate hematopoietic stem
cells, said method comprising combining isolated primate
hematopoietic stem cells which can undergo replication in culture
with a recombinant retrovirus comprising a genome based on a
retroviral vector into which a gene X is inserted, wherein said
combining uses means whereby said hematopoietic stem cells and said
recombinant retrovims are brought into close physical contact, so
that chance of infection of said hematopoietic stem cells by said
recombinant retrovirus is significantly enhanced.
36. The method according to claim 35, wherein said replication is
stimulated by adding recombinant hematopoietic growth factors to
said culture.
37. The method according to claim 35, wherein said culture includes
a stromal cell support and replication is stimulated by
hematopoietic growth factors produced by said stromal cell
support.
38. The method according to claim 35, wherein said combining is
cocultivating with a cell line which produces said recombinafit
retrovirus and wherein said replication is stimulated by
hematopoietic growth factors produced by said cell line.
39. A cell culture comprising: isolated primate hematopoietic stem
cells which can undergo replication and a retrovims producing cell
line which produces a recombinant retrovirus comprising a genome
based on a retroviral vector into which a gene X is inserted.
40. The cell culture of claim 39, further comprising a stromal
cell.
41. The method according to claim 5, wherein said compound which
has binding affinity for both hematopoietic stem cells and said
recombinant retrovirus is a component present in a natural
hematopoietic environment.
42. The method according to claim 5, wherein said compound which
has binding affinity for both hematopoietic stem cells and said
recombinant retrovirus is a component of cells derived from a
natural hematopoietic environment.
43. A method for genetically modifying primate hematopoietic stem
cells, said method comprising: combining isolated primate
hematopoietic stem cells which can undergo replication with a
recombinant retrovirus comprising a genome based on a retroviral
vector into which a gene X is inserted, wherein said combining uses
means whereby said hematopoietic stem cells and said recombinant
retrovirus are brought into close physical contact, so that chance
of infection of said hematopoietic stem cells by said recombinant
retrovirus is significantly enhanced.
44. A cell culture comprising: isolated primate hematopoietic stem
cells which can undergo replication and a stromal cell population,
wherein said stromal cell population comprises cultured cells,
extracellular matrix molecules and cytokines produced by said
cultured cells.
45. The cell culture according to claim 44, wherein said stromal
cell population is derived from human bone marrow mononuclear
cells.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
08/820,479, filed Mar. 18, 1997, pending, now U.S. Pat. No. ______,
which is a continuation-in-part of application Ser. No. 08/211,342,
filed Mar. 24, 1994, now U.S. Pat. No. 5,612,206 issued Mar. 18,
1997, the contents of which is incorporated herein by
reference.
TECHNICAL FIELD
[0002] 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 such a method. The method is exemplified by the use
of means which enhance the local concentration of retroviral
particles derived from the murine leukemia virus in the vicinity of
target primate stem cells.
BACKGROUND
[0003] Developments in the field of molecular biology have led 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 with the diseases that occur most
frequently will have been identified, cloned and characterized
before the end of this century.
[0004] So far, molecular genetics has contributed to medicine by
the development of diagnostic 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 treatment,
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 be one of the
possibilities. Although this field of study is still in a
preliminary stage and must be developed, therapeutic possibilities
are in the distance which can drastically improve medicine in the
future (Anderson, Science 226:410 (1984); Belmont and Caskey, in
Gene Transfer, R. Kucherlapati, eds, Plenum press, New York and
London, p. 411 (1986); and Williamson, Nature 298:416 (1982)).
[0005] 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
phenomenon 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, for
example, collected perinatally from the umbilical vein, are useful
sources for stem cells. The underlying idea of a gene therapy
directed to hematopoietic stem cells is that gene transfer to (a
limited number of) stem cells may already be sufficient to replace
the entire blood-forming tissue with genetically modified cells for
a lifetime (Williamson, supra). 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 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.
[0006] 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 herein, 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 themselves to cause a productive infection because
the retroviruses used are replication-defective and are not
contaminated with wild-type viruses (Temin, Gene Transfer,
supra).
[0007] 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. The viruses include, but are not restricted to, murine
leukemia viruses (MuLV; Weiss et al., RNA Tumor Viruses, New York
(1984)) with a so-called amphotropic or xenotropic host-range,
gibbon ape leukemia viruses (GaLV; Lieber et al., Proc. Natl. Acad.
Sci. USA 72, 2315-2319 (1975)), and primate lentiviruses.
[0008] For the production of recombinant retroviruses, two elements
are required: the so-called retroviral vector, which, in 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 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, Hum. Gene Ther., 1:5 (1990)).
[0009] As the presence of replication-competent viruses in a gene
therapy protocol is considered highly undesirable, most modem
packaging cell lines are constructed in a way such that the risk 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 introducing them into the cell line separately (Danos
and Mulligan, Proc. Natl. Acad. Sci. USA 85:6460 (1988); Markowitz
et al., J. Virol. 62:1120 (1988); and Markowitz et al., Virology
167:400 (1988)).
[0010] 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 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 .psi.CRIP (Danos & Mulligan,
Proc. Natl. Acad. Sci. USA 85:6460 (1988)) 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 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 .psi.CRIP cells (Van Beusechem
et al., J. Exp. Med. 172:729 (1990)) which on account of its
instability 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.
[0011] Studies in mice have demonstrated that using amphotropic
retroviral vectors, bone 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 Beusechem et al., supra).
[0012] Previous problems with regard to the nonexpression of the
newly introduced genes were 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 Beusechem
et al., supra; and Valerio et al., Gene 84:419 (1989)). In the
present invention, these vectors are called LgXL
(.DELTA.Mo+PyF101), wherein X represents the code of a gene yet to
be filled in.
[0013] 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 efficient gene
transfer is also possible to blood-forming stem cells of higher
mammals, in particular primates. Moreover, genetic modification
coupled with autologous bone 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.
[0014] The experimental animal model is eminently suitable for
preclinical gene therapy studies is the non-human 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 bone 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 autologously (i.e., into the
same monkey) after the endogenous bone marrow cells have been
eradicated by irradiation.
[0015] To date, such experiments have met with little success with
regard to:
[0016] a) the hematopoietic regeneration that could be effected
with the infected bone marrow, and
[0017] b) the in vivo stability of the genetic modification.
[0018] In the studies published to date, gene transfer was
performed either by incubating bone 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 co-cultivation
wherein the virus-producing cells are adhered to the bottom of a
culture bottle and the bone marrow cells are seeded on top thereof.
Following co-cultivation, the non-adherent bone marrow cells are
subsequently harvested and used as transplants.
[0019] In the first reported study (Anderson et al., "Gene transfer
and expression in non-human primates using retroviral vectors," in
Cold Spring Harbor Symposia on Quantitative Biology, Volume LI,
eds. Cold Spring Harbor Laboratory, New York, p. 1073 (1986); and
Kantoff, P. W., A. P. Gillio, J. R. McLachlin, C. Bordignon, et
al., J. Exp. Med. 166:219 (1987)), in 19 monkeys an autologous
transplantation was performed with bone marrow cells infected with
different retroviral vectors containing the gene for neomycin
resistance (neo.sup.r) or dihydrofolate reductase (DHFR), or with a
virus in which neo.sup.r 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 co-cultivation procedure and the infection with
virus supernatant that can be harvested from the virus-producing
cells were utilized. Using the co-cultivation 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 marrow and in four of these animals the
gene could be demonstrated. 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.
[0020] In the second study (Bodine et al., Proc. Natl. Acad. Sci.
USA 87:3738 (1990)), bone marrow from rhesus monkeys was
co-cultivated with cell lines that produce neo.sup.r-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, no long-term studies could be performed because again the
bone marrow proved incapable of reconstituting the hematopoietic
system.
[0021] In conclusion, in the data published so far, the
co-cultivation method has always been associated with a drastic
loss of in vivo regenerating capacity of the bone marrow cells
(Anderson et al., "Gene transfer and expression in non-human
primates using retroviral vectors," in Cold Spring Harbor Symposia
on Quantitative Biology, Volume LI, eds. Cold Spring Harbor
Laboratory, New York, p. 1073 (1986); Kantoff, P. W., A. P. Gillio,
J. R. McLachlin, C. Bordignon, et al., J. Exp. Med. 166:219 (1987);
and Bodine et al., supra), so that a clinical application is
precluded.
[0022] In addition, none of the studies published to date are
sufficiently 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.
[0023] Relevant Literature
[0024] Fibronectin as a single molecule has been reported to bind
retroviruses and hemopoietic cells, thereby enhancing the gene
transfer efficiency (WO 95/26200).
BRIEF SUMMARY OF THE INVENTION
[0025] 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, 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 a retroviral vector which is
derived from 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 an
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 close physical contact without departing from the
present invention.
[0026] The term "hematopoietic stem cell" is understood to mean a
cell that has the following characteristics: (1) it has the ability
to differentiate into any type of cell of the blood cell system,
and (2) it has the capacity to multiply itself without losing its
characteristics 1 and 2. The term "hematopoietic cell" is
understood to 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 concentration of retrovirus
particles in the direct vicinity of the target cell beyond that
obtained under standard conditions, where "standard conditions" are
those where retrovirus particles and target cells are mixed
together in a liquid solution at normal gravitation.
[0027] In one embodiment of the invention, isolated hematopoietic
cells from a primate are, by means of a co-cultivation, exposed to
cells that produce the recombinant retrovirus. During this
co-cultivation, isolated hematopoietic cells are in the direct
vicinity of virus-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
virus-producing cells. During the co-cultivation, virus-producing
cells continuously produce new recombinant retroviruses that are
shed from the cell membrane into the culture medium. After their
production, recombinant retroviruses have a limited life span that
depends at least in part on their nature, on the culture
temperature and on the composition of the culture medium. Hence,
the shorter the distance recombinant retroviruses have to travel
from the site where they were shed into the medium towards the
isolated hematopoietic cells from a primate the higher is the
chance for a successful genetic modification of isolated
hematopoietic cells of a primate. In this aspect of the invention,
therefore, the most efficient genetic modification is obtained for
the subset of isolated hematopoietic cells from a primate that most
intimately adhere to virus-producing cells. For this reason, it is
preferred that following co-cultivation both nonadherent and
adherent cells are harvested.
[0028] In another embodiment, the intimate interaction between
virus-producing cells and 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 the mixture on the same
physical site by electrodiffusion, forcing by pressure or
centrifugation the 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 the mixture. In the latter
application of the invention, the pressure is either positive
pressure applied to the fluid medium or negative pressure applied
to the porous solid material or to a space past the porous solid
material. Alternatively, 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.
[0029] In yet another embodiment, 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 hematopoietic stem cell and recombinant retrovirus.
The compound is characterized by its capacity to bind (1) the
hematopoietic stem cell, and (2) the recombinant retrovirus and/or
the virus-producing cell. It is preferred that the compound besides
binding to the recombinant retrovirus or virus-producing cell
preferentially, or even exclusively, binds to the hematopoietic
stem cell. In this way, the compound selectively increases the
genetic modification of the hematopoietic stem cell. The 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. The 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. The hematopoietic stem cells by nature closely
interact with cells and extracellular matrix molecules present in
the hematopoietic microenvironment. In addition, the cells produce
cytokines that support the maintenance and functioning of the
hematopoietic stem cells and the extracellular matrix molecules
bind cytokines that support the maintenance and functioning of the
hematopoietic stem cells.
[0030] The method of the invention is also performed using a
different kind of compound that (1) binds to the recombinant
retrovirus vector and (2) is immobilized on a solid support
material. The hematopoietic cells of a primate are brought in close
contact with the solid support material (and thereby with the bound
recombinant retrovirus vector) by any other means exemplified in
the various embodiments of the invention (like gravity,
electrodiffusion, and fluid flow).
[0031] In another embodiment of the invention the close contact
between the recombinant retrovirus and the hematopoietic stem cell
is accomplished by forcing the recombinant retrovirus towards the
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 towards the hematopoietic stem cell
by electrodiffusion, and forcing the recombinant
retrovirus-containing medium through a bed of hematopoietic cells
including the hematopoietic stem cell. In the latter case, the
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 the cells to pass. In this
application of the invention, force is provided by either normal
gravity, increased gravity through centrifugation, positive
pressure applied to the medium, or negative pressure applied to the
porous solid material or to a space past the porous solid material.
In this aspect of the invention it is preferred but not essential
that the solid material used binds the recombinant retrovirus.
[0032] As is clear from the foregoing, the invention provides means
to bring isolated hematopoietic cells including hematopoietic stem
cells from a primate in close physical contact with a recombinant
retrovirus. This is accomplished either directly, by bringing the
recombinant retrovirus itself in close proximity of the isolated
hematopoietic cells, or indirectly, by bringing cells that produce
the recombinant retrovirus in close proximity of the isolated
hematopoietic cells.
[0033] According to the invention, it is preferred that the
retroviral vector comprises two LTRs long terminal repeats) derived
from a viral MuLV vector and the 5' part of the gag gene of a MuLV.
The 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 enhancer instead of the
Mo-MuLV enhancer. To this end, preferably the retroviral vector
pLgXL(.DELTA.Mo+PyF101) is used, wherein X represents the genetic
information to be introduced into the bone marrow cells.
[0034] According to the invention, producer cells that can be used
include all recombinant 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(.DELTA.Mo+PyF101) structure useful in the invention have been
disclosed in PCT International Patent Application WO96/35798. 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
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+envAM 12
cells are preferred, it is further preferred that the cells that
produce a recombinant retrovirus contain several copies of the
retroviral vector.
[0035] 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 the hematopoietic stem cells for a period of
time in the absence of recombinant retrovirus and virus-producing
cells before being subjected to genetic modification with
recombinant retrovirus or to culture the hematopoietic 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.
[0036] 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
[0037] The invention provides a method for introducing a gene X
into isolated hematopoietic cells including hematopoietic stem
cells from a primate, whereby the isolated hematopoietic cells are
brought in close contact with a recombinant retrovirus. Preferably,
the recombinant retrovirus is an amphotropic retrovirus whose
genome is composed of the recombinant retroviral vector
pLgXL(.DELTA.Mo+PyF101) 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(.DELTA.Mo+PyF101), a virus-producing cell
line shedding recombinant pLgXL(AMo+PyF101) retrovirus, and a
method by which isolated hematopoietic cells or purified
hematopoietic stem cells of a primate are provided with gene X.
[0038] Hematopoietic Cells
[0039] Many different standard procedures are known in the art for
the collection, storage, processing, and reinfusion of hemopoietic
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 supportive care (see, e.g.,
Bone Marrow and Stem Cell Processing: A Manual of Current
Techniques, eds. E. M. Areman, H. J. Deeg, and R. A. Sacher, F. A.
Davis Company, Philadelphia, pp. 487 (1992); Marrow
Transplantation: Practical and Technical Aspects of Stem Cell
Reconstitution, eds. R. A. Sacher and J. P. AuBuchon, American
Association of Blood Banks, Bethesda, Md., pp. 187 (1992)). 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 purity and recovery, and is thus
preferred.
[0040] True in vitro tests for hemopoietic 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 (Knan-Shanzer et al., Gene Therapy, 3:323-333 (1996)). In
the experiments with rhesus monkeys described in the examples this
was not done, because not all essential antibodies for this
analysis react with rhesus monkey cells. There is no special
treatment of the cells prior to cultivation with retrovirus
particles.
[0041] Recombinant Retroviral Vector pLgXL(.DELTA.Mo+PyF101)
[0042] The recombinant retroviral vector includes DNA elements
originating from a MuLV which are necessary in cis for the
packaging, 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 originating from MuLV with
the enhancer of the polyoma virus strain PyF101 (Linney et al.,
Nature 308:470 (1984)). 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
Beusechemetal., J. Exp. Med. 172:729 (1990); and Valerio et al.,
Gene 84:419 (1989)), 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,
"Effect of internal viral sequences on the utility of retroviral
vectors," J. Virol. 61:1647 (1987)), 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 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(.DELTA.Mo+PyF101) vector meets these
requirements and includes some further improvements, as exemplified
above.
[0043] 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, Gene
2:95 (1977)) or a vector from the pUC series (Vieira and Messing,
Gene, 19:259(1977)); on these, both an origin of replication and a
selectable gene (for instance for ampicillin to be of tetracycline
resistance) are present, together with gene X. The term "gene" is
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 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 of Autosomal Dominant
Autosomal Recessive, and X-Linked Phenotypes, 8.sup.th edition,
John Hopkins University Press (1988); and Stanbury et al., The
Metabolic Basis of Inherited Disease, 5th edition, McGraw-Hill
(1983).
[0044] Examples of gene X include: genes associated with diseases
of the carbohydrate metabolism such as for: fructose-1-phosphate
aldolase; fructose-1,6-diphosphatase; glucose-6-phosphatase;
lysosomal .alpha.-1,4-glucosidase; amylo-1,6-glucosidase;
amylo-(1,4:1,6)-transgluc- osidase; muscular phosphorylase; liver
phosphorylase; muscular phosphofructokinase;
phosphorylase-b-kinase; galactose-1-phosphate uridyl transferase;
galactokinase; all enzymes of the pyruvate dehydrogenase complex;
pyruvate carboxylase; 2-oxoglutarate glyoxylate carboligase; and
D-glycerate dehydrogenase; genes associated with diseases of the
amino acid metabolism such as for: phenylalanine hydroxylase;
dihydrobiopterin synthetase; tyrosine aminotransferase; tyrosinase;
histidase; fumarylacetoacetase; glutathione synthetase;
.gamma.-glutamylcysteine synthetase;
orinithine-.delta.-aminotransferase; carbamoylphosphate synthetase;
ornithine carbamyltransferase; argininosuccinate synthetase;
argininosuccinate lyase; arginase; L-lysine dehydrogenase; L-lysine
ketoglutarate reductase; valine transaminase; leucine isoleucine
transaminase; "branched chain" 2-keto acid decarboxylase;
isovaleryl CoA dehydrogenase; acyl-CoA dehydrogenase;
3-hydroxy-3-methylglutaryl CoA lyase; acetoacetyl CoA
3-ketothiolase; propionyl CoA carboxylase; methylmalonyl CoA
mutase; ATP:cobalamine adenosyltransferase; dihydrofolate
reductase; methylene tetrahydrofolate reductase; cystathionine
.beta.-synthase; sarcosine dehydrogenase complex; proteins
belonging to the glycine cleavage system; .beta.-alanine
transaminase; serum carnosinase; and cerebral homocarnosinase;
genes associated with diseases of fat and fatty acid metabolism
such as for: lipoprotein lipase; apolipoprotein C-II;
apolipoprotein E; other apolipoproteins; lecithin cholesterol
acyltransferase; LDL receptor; liver sterol hydroxylase; and
"Phytanic acid" .alpha.-hydroxylase; genes associated with
lysosomal defects such as for: lysosomal .alpha.-L-iduronidase;
lysosomal iduronate sulfatase; lysosomal heparin N-sulfatase;
lysosomal-acetyl-.alpha.-D-sulfatase; lysosomal acetyl
CoA:.alpha.-glucosaminide N-acetyltransferase; lysosomal
N-acetyl-.alpha.-D-glucosaminide 6-sulphatase; lysosomal
galactosamine 6-sulphate sulfatase; lysosomal .beta.-galactosidase;
lysosomal arylsulfatase B; lysosomal .beta.-glucuronidase;
N-acetylglucosaminylphos- photransferase; lysosomal
.alpha.-D-mannosidase; lysosomal .alpha.-neuraminidase; lysosomal
aspartylglycosaminidase; lysosomal .alpha.-L-fucosidase; lysosomal
acid lipase; lysosomal acid ceramidase; lysosomal sphingomyelinase;
lysosomal glucocerebrosidase; lysosomal galactosylceramidase;
lysosomal arylsulfatase A; .alpha.-galactosidase A; lysosomal acid
.beta.-galactosidase; and .alpha.-chain of the lysosomal
hexosaminidase A; genes associated with diseases of the steroid
metabolism such as for: 21-hydroxylase; 11.beta.-hydroxylase;
androgen receptor; steroid 5.alpha.-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; genes associated with diseases of the
porphyrin and heme metabolism such as for: uroporphyrinogene III
cosynthase; ferrochelatase; porphobilinogene deaminase;
coproporphyrinogene oxidase; proporphyrinogene oxidase;
uroporphyrinogene ill synthase; uroporphyrinogene decarboxylase;
bilirubin UDP-glucuronyltransferase; and catalase; genes associated
with diseases of the connective tissue, muscles and bone such as
for: lysyl hydroxylase; procollagen peptidase;
.alpha.1-antitrypsin; dystrophin; alkaline phosphatase; and guano
sine 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
coagulation factor XIII; all other blood coagulation factors; all
genes associated with osteopetrosis such as for: "carbonic
anhydrase II"; thrombocytes membrane glycoprotein lb; thrombocytes
membrane glycoprotein IIb-IIIa; spectrin; pyruvate kinase;
glucose-6-phosphate dehydrogenase; NADH cytochrome
b.sub.5reductase; .beta.-globin; and .alpha.-globin; genes
associated with diseases of transport systems such as for: lactase;
sucrase-.alpha.-dextrinase; 25-hydroxyvitamin
D.sub.3-1-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 C10; the inhibitor of Cl, a component of the
complement system; the inactivator of C3b, a component of the
complement system; 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; genes coding for hormones as well as the genes
coding for their receptors such as for instance for growth
hormone.
[0045] Gene X also includes genes which (to date) have not been
associated with a hereditary defect but with which gene therapy can
be practiced in some manner. These include: the gene for tyrosine
hydroxylase, drug resistance genes such as for instance: the
P-glycoprotein P170 (the so-called multidrug resistance gene mdr
1); 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 interleukins and all
interferons; genes coding for all growth factors; genes coding for
all growth factor receptors; 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
(Sullenger et al., Cell 63:601 (1990)), 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 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 (Borelli et al., Proc. Natl. Acad. Sci.
USA 85:7572 (1988); and Mansour et al., Nature 336:348 (1988)).
[0046] The Virus-Producing Cells
[0047] In order to obtain a stable, selectable virus-producing cell
line which produces the amphotropic recombinant retrovirus,
pLgXL(.DELTA.Mo+PyF101) is introduced into an amphotropic packaging
cell line that is selected 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.,
Virology, 167:400 (1988)). It has been demonstrated, on the other
hand, that .psi.CRIP is not selectable and is unstable with respect
to virus production (Danos and Mulligan, Proc. Natl. Acad. Sci. USA
85:6460 (1988)).
[0048] The selectable packaging cell line is based on mammalian
cells and produces all viral proteins that are coded by the gag,
pol and env genes of MuLV. The env gene must originate from a virus
with a tropism including 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 a RNA polymerase 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, Mol. Cell.
Biol. 6:2895 (1986)), but the genes may also occur separately on
two expression vectors as described by Markowitz (Markowitz et al.,
Virology 167:400 (1988)). This last is to be preferred because it
reduces the chances of recombination events leading to helper virus
formation.
[0049] As stated, a useful characteristic of the packaging cell
line to be used for this invention is the possibility it provides
means of selecting for the presence of the above-mentioned
packaging constructs. This can be achieved by effecting a physical
association of the packaging constructs with a selectable marker
gene. This association can be achieved by combining them in one
vector (as done with pGag-Po1GPT in reference (Markowitz et al., J.
Virol. 62:1120 (1988)) or by means of a so-called cotransfection
(review in for instance (Pellicer et al., Science 209:1414 (1980)).
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 in the
genome of the transfected cell (Pellicer et al., Science 209:1414
(1980)), the thus selected cells will mostly contain the packaging
construct as well. In view of the fact that .psi.CRIP 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.
[0050] As a marker gene, genes coding for a large number of
different proteins can be used. Widely used and preferred marker
genes are: the neomycin resistance gene (Southern and Berg, J. Mol.
Appl. Genet. 1:327 (1982)), the hygromycin resistance gene
(Blochlinger and Diggelman, Mol. Cell. Biol. 4:2929(1984)), the E.
coli xanthine-guaninephosphoribosyl transferase (gpt) gene
(Mulligan and Berg, Science 209:1422 (2980)), the histidinol gene
(Hartman and Mulligan, Proc. Natl. Acad. Sci. USA 85:8047 (1988)),
the herpes simplex virus thymidine kinase gene (Colbre-Garapin et
al., Proc. Natl. Acad. Sci. USA 76:3755 (1979)) and the
methotrexate-resistant isotype of dihydrofolate reductase (Simonsen
and Levinson, Proc. Natl. Acad. Sci. USA 80:2494(1983)). These
genes must also be under control of a suitable promoter, in
particular a RNA polymerase II promoter, and be followed by a
polyadenylation signal.
[0051] The introduction of pLgXL(.DELTA.Mo+PyF101) can be effected
by means of various physical techniques such as calcium-phosphate
precipitation, electroporation or lipofection (Graham and Van der
Eb, Nucl. Acids Res. 15:1311 (1973); Potter et al., Proc. Natl.
Acad. Sci. USA 81:7161(1984); Felgner and Ringold, Nature
337:387(1989); Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413
(1987)). If the packaging cells cannot be selected for the presence
of pLgXL(.DELTA.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(.DELTA.Mo+PyF101).
The successfully transfected cells are then be selected by
selecting for the marker gene. Since the DNA fragments mostly end
up ligated to each other in one place in the genome of the
transfected cell, the thus selected cells will mostly contain the
retroviral vector as well.
[0052] A preferred procedure is the introduction of
pLgXL(.DELTA.Mo+PyF101) via an infection. Since viruses are not
capable of infecting packaging cells of the same tropism, use must
be 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 a different tropism.
For example, ecotropic virus produced by ecotropic packaging cells
transfected with a pLgXL(.DELTA.Mo+PYF101) construct can be used to
infect amphotropic packaging cells. The infected cells are cloned
and then tested for their ability to produce virus.
[0053] 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 (Bestwick et al., Proc. Natl. Acad. Sci. USA
85:5404 (1988); and Kozak and Kabat, J. Virol. 64:3500 (1990)). In
this method, an ecotropic virus-producing cell line is
co-cultivated with amphotropic packaging cells, which can give rise
to repeated infections. In order to enable the amphotropic cells to
be cloned back after this co-cultivation, they must 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.
[0054] Method by Which Hematopoietic Cells of a Primate Can Be
Provided with Gene X. In Such a Manner That the Regeneration
Capacity of the Hematopoietic Cells is Maintained and Autologous
Transplantation of the Hematopoietic Cells Gives Rise to a
Genetically Modified Hematopoietic System
[0055] The above-mentioned recombinant retroviral vectors are used
for the efficient introduction of gene X into hematopoietic cells
of primates by bringing the hematopoietic cells in close physical
contact with the 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 recombinant
retroviral vector particle to associate with its receptor on the
surface of a hernatopoietic 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.
[0056] 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 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 the target
cell according to one of several means which are exemplified in
detail below.
[0057] 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 for 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.
[0058] 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, 19-28
(1994); Forestell, et al., Gene Ther. 2, 723-730 (1995)). This
half-life can be increased by lowering the culture temperature to
32.degree. C. (Kotani et al., Hum. Gene Ther. 5, 19-28 (1994)).
Methods to increase the stability of the recombinant retroviral
vector particles are not part of the invention. It is realized,
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.
[0059] It is of critical importance that a method to transfer a
gene X into a certain target cell allows the target cell to retain
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 on bone
marrow grafts ofprimates led to a dramatic loss of the in vivo
regenerating capacity of the grafts (see 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.
[0060] Numerous different procedures for harvest, processing,
storage, shipping, etc. of human hemopoietic cells are in use and
known to those of skill in the art. Various means of bringing
together of the recombinant retrovirus particles and hematopoietic
target cells include the following:
[0061] General Procedure for Recombinant Retroviral-Vector Mediated
Gene Transfer into Hematopoietic Cells of Primates:
[0062] The hematopoietic cells of a primate are suspended in a
suitable culture medium for hematopoietic cells containing
recombinant retrovirus vector particles. The 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 positive selection for CD34+cells (FACS sorting, Dynabeads
immunoselection, Miltenyi MACS selection, AIS CELLector flask
selection, or CellPro CEPRATE selection) or depletion of cells
carrying mature cell type cell surface markers. Many different
suitable culture media are commercially available. They include,
but are not restricted to DMEM, IMDM, and A-MEM, with 5-30% serum
and often further supplemented with, for example, BSA, one or more
antibiotics. 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 HCl are added. Usually, the cultures are maintained for
2-4 days and the recombinant retrovirus vector containing medium is
refreshed daily. Optionally, the hematopoietic 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.
[0063] For successful gene transfer it is essential that the target
cells undergo replication in culture (without differentiation).
Most harvested hemopoietic stem cells are resting cells. Therefore,
a stimulus to enter cell cycle during culture is needed. This can
be accomplished by adding recombinant hemopoietic 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.
[0064] 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.
[0065] The following are examples of indirect methods which are
used.
[0066] i) Co-cultivation of hematopoietic cells of a primate with
virus-producing cells. The virus-producing cells and the
hematopoietic cells from a primate are mixed at the initiation of
the co-culture, or a monolayer of adherent virus-producing cells is
established before adding the hematopoietic cells from a primate.
The virus-producing cells may have been damaged prior to initiation
of the co-culture by, for example, a lethal dose of irradiation,
but can be used so long as they continue to shed recombinant
retroviral vector particles into the medium. The virus-producing
cells have the capacity to bind the hematopoietic cells to their
surface. Virus-producing cells based on the commonly used packaging
cells derived from mouse fibroblasts have this capacity. Due to the
intimate interaction between the virus-producing cell and the
hematopoietic cell the recombinant retroviral vectors produced by
the virus-producing cell only have to travel a very short distance
to reach the 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 destabilizing 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 virus-producing cells preferentially, or even exclusively,
bind hematopoietic stem cells. In this way, the genetic
modification of hematopoietic stem cells is selectively increased.
In this embodiment of the invention, it is preferred that the
co-cultivation 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 (IL-3). Following co-cultivation, both the
non-adherent and the adherent cells are harvested from the culture
(the last-mentioned cells can be obtained by, for example,
trypsinization) and used as the transplant.
[0067] ii) Co-cultivation of hematopoietic cells of a primate with
virus-producing cells at increased gravitational force. This
embodiment of the invention provides a further improvement of and
includes the advantages 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
effects 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
prevented from traveling away from the hematopoietic cells, thus
increasing the local concentration of the particles. The increased
gravitational force is achieved by performing the co-cultivation
while spinning the container with the culture around an axis of
rotation. The axis can intersect the container or be located
outside of the container. Useful centrifuges to spin the cultures
according to the invention are known in the art. The gravitational
force is maximized, but should not exceed the maximal gravitational
force that allows functional survival of the virus-producing cells,
the recombinant retroviral vector particles and the hematopoietic
cells from a primate. Usually, the gravitational force will not
exceed 2500 g. As a result of the further increased gene transfer
efficiency obtained with this embodiment of the invention, the
co-culture duration can be significantly shortened. Usually, this
procedure will not be performed for more than eight consecutive
hours.
[0068] iii) Co-cultivation 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
electrodiffusion, the procedure is performed as described under
(i). Because the hematopoietic 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 co-cultivation
procedure in an electrophoresis unit, 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 hematopoietic 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 the particles. Electrophoresis units
useful for this aspect of the invention are known in the art. The
electrophoresis unit preferably contains two chambers separated by
a semi-permeable membrane, with pore sizes that do not permit
passage of cells and vectors. In such a two-chamber electrophoresis
unit, co-cultivation 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
the hematopoietic cells of a primate, the virus-producing cells,
and the recombinant retroviral vector particles. To further reduce
damage, voltage may be applied periodically. Also in this
embodiment of the invention, virus-producing cells and
hematopoietic cells from a primate are mixed at the initiation of
the co-culture, or a monolayer of adherent virus-producing cells is
established on the surface of the semi-permeable membrane before
adding the hematopoietic cells from a primate.
[0069] iv) Co-cultivation of hematopoietic cells of a primate with
virus-producing 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 described under (i). Apart from the
fluid flow, the procedure is performed as described under (i). The
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 the culture medium but small enough in size to prevent
passage of the hematopoietic cells of a primate and the
virus-producing cells. The pores may or may not allow passage of
the recombinant retroviral vectors. The force driving the fluid
flow is exercised by normal or increased gravitational force or by
positive pressure applied to the culture medium or by negative
pressure applied to the porous solid material or to a space past
the porous solid material. The increased gravitational force is
achieved by performing the co-cultivation while spinning the
container with the culture around an axis of rotation. The axis can
intersect the container or be located outside of the container. The
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 the 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 the pores do not allow passage of the recombinant
retroviral vector the rate is maximized; but in no case may the
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, the virus-producing cells and the hematopoietic
cells from a primate are mixed at the initiation of the co-culture,
or a monolayer ofadherent virus-producing cells is established on
the surface of the porous solid material before adding the
hematopoietic cells from a primate.
[0070] v) Co-cultivation of hematopoietic cells of a primate with
virus-producing cells in the presence of a compound that binds both
the hematopoietic cells of a primate and the virus-producing cells.
This embodiment of the invention provides another alternative
improvement of and includes the advantages of the procedure
described under (i). Apart from the compound, the procedure is
performed as described under (i). The compound has at least one
binding site for the hematopoietic cell of a primate and at least
one binding site for the virus-producing cell. The nature of the
binding sites may be different or the same. The 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. The 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 even via a
complex mixture of different cells. The mixture of cells may be
artificially composed or be derived from naturally occurring cell
mixtures or tissues. Thus, it is to be understood that the
compound, may, for example, 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 the hematopoietic cells of a primate and the
virus-producing cells. In this embodiment of the invention it is
preferred that the binding site for a hematopoietic cell of a
primate has a binding preference for hematopoietic stem cells over
other types of hematopoietic cells.
[0071] Preferred compounds in this aspect of the invention are
derived from or are components of the natural hematopoietic
microenvironment present in the bone marrow of animals. The
hematopoietic stem cells by nature closely interact with cells and
extracellular matrix molecules present in the hematopoietic
microenvironment. In addition, the cells produce cytokines that
support the maintenance and functioning of the hematopoietic stem
cells and the extracellular matrix molecules bind cytokines that
support the maintenance and functioning of the hematopoietic stem
cells. We are using a cultured stroma cell population. This is a
complex 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 extracellular matrix include collagens,
proteoglycans, fibronectin, laminin, elastin, glycosaminoglycans,
thrombospondin, and chondronectin.
[0072] Further preferred compounds in this aspect of the invention
comprise parts that are derived from antibodies or from peptides
with a defined binding capacity. The 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 the natural processes
involve at least one cell type of the hematopoietic system.
[0073] 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, for example, peptides
derived from cytokines known to act on the hematopoietic cells by
binding to a specific receptor molecule).
[0074] 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
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.
[0075] vi) Sedimentation of recombinant retrovirus vectors onto
hematopoietic cells of a primate at increased gravitational force.
The increased gravitational force is achieved by incubating the
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). The gravitational force should at least be higher than the
minimal force needed to overcome the random diffusion of the
recombinant retroviral vector 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, the gravitational force will not exceed 2500 g.
The centrifugation time depends on the centrifugation speed and on
the height of the column of culture medium above the 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.
[0076] vii) Electrodiffusion ofrecombinant retroviral vectors
towards hematopoietic cells of a primate. By performing the
cultivation of the hematopoietic cells of a primate in recombinant
retroviral vector containing medium in an electrophoresis unit the
hematopoietic cells of a primate and recombinant retroviral vector
particles that are both negatively charged are forced to move in
the same direction towards the positive electrode and thus are
concentrated. Electrophoresis units useful for this aspect of the
invention are known in the art. The electrophoresis unit preferably
contains two chambers separated by a semi-permeable membrane, with
pore sizes that do not permit passage of the hematopoietic cells of
a primate and the recombinant retroviral vectors. In such a
two-chamber electrophoresis unit the cultivation 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 the hematopoietic cells of a primate
and the recombinant retroviral vector particles. To further reduce
the damage, the voltage maybe 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 containing medium.
[0077] viii) Forcing recombinant retroviral vector particles
towards hematopoietic cells of a primate by fluid flow. The 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 passage of the culture medium
but small enough in size to prevent passage of the hematopoietic
cells of a primate. The pores may or may not allow passage of the
recombinant retroviral vectors. The force driving the 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 primate. The time
during which the fluid flow is maintained typically does not exceed
two recombinant retroviral vector half-lives. Optionally, the
procedure may be repeated several times with fresh recombinant
retroviral vector containing culture medium.
[0078] ix) Culture of hematopoietic cells of a primate in
recombinant retroviral vector containing medium in the presence of
a compound that binds both the hematopoietic cells of a primate and
the recombinant retroviral vector. The compound has at least one
binding site for the hematopoietic cell of a primate and at least
one binding site for the recombinant retroviral vector. The nature
of the binding sites may be different or the same. The 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 the binding site for a hematopoietic
cell of a primate has a binding preference for hematopoietic stem
cells over other types of hematopoietic cells.
[0079] 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
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 the vector and cell, or with a mixture of synthetic
peptides directed against the vector (e.g., peptides derived from
the receptor for the retrovirus envelope molecule) and the
cell.
[0080] x) Binding the recombinant retroviral vector to a compound
that is immobilized on a solid support material that is brought 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 the recombinant retroviral vector
while being immobilized to the solid support material by any
physical or chemical means. The 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), DEAE-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 support material)
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 multi-layer tissue culture dish or
flask, semipermeable membrane, porous or nonporous beads including
immunomagnetic beads, and (hollow) semipermeable or nonpermeable
fibers. Methods of coating and coupling the compound to the solid
support materials include the following.
[0081] For polystyrene a simple adsorption procedure can be
followed. Protein dissolved in PBS is incubated for several hours
at room temperature with the solid support material. Subsequently,
the coated solid support material is washed once or several times
in PBS or in PBS with 0.1% (w/v) irrelevant protein such as, for
example, albumin.
[0082] 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, for example,
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
buffer at high pH (preferably in the range of 8-10) with a high
salt content (preferably approximately 0.5M NaCl) for 2 hours at
room temperature or for 10-16 hours at 4.degree. C. Subsequently,
excess ligand is washed away with coupling buffer, any remaining
active groups are blocked with, for example, 0.1 M Tris-HCl buffer
pH 8.0 for 2 hours at room temperature or for 10-16 hours at
4.degree. C., and ionically bound free ligand is washed away by
alternatively washing with high and low pH buffer solutions such
as, for example, Tris-HCl buffer pH 8.0 with 0.5M NaCl and 0.1M
acetate buffer pH 4.0 with 0.5M NaCl. Another example is
polystyrene activated by p-toluenesulfonyl chloride treatment (such
as the Tosylactivated Dynabeads M-450 manufactured by Dynal). Any
protein or glycoprotein can be chemically coupled to this material
by incubating the solid support material with the ligand dissolved
in a high pH buffer such as 0.5M borate buffer pH 9.5 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 the invention are
known in the art (see, for example, Affinity Chromatograph. A
Practical Approach, eds. P. D. G. Dean, W. S. Johnson, and F. A.
Middle, IRL Press, Oxford, pp. 215 (1985)). 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.
[0083] Finally, natural intermolecular interactions can be
exploited to couple proteins to a solid support material, for
example, peptides containing a histidine-tag efficiently interact
with materials containing nickel ions.
[0084] The recombinant retroviral vector is bound to the compound
by incubating a preparation of the recombinant retroviral vector in
the tissue culture dish and the hematopoietic cells of a primate
are brought in close contact with the recombinant retrovirus by
seeding the hematopoietic cells of a primate in the tissue culture
dish, where the contact maybe further enhanced by, for example,
increasing the gravitational force.
[0085] 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, 72:191-195(1988); Sutherland
et al., Blood, 74:1563-1570(1989); Breems et al., Leukemia,
8:1095-1104 (1994)). Furthermore, a candidate stem cell population
can be analyzed phenotypically (Knan-Shanzer et al., Gene Therapy,
3:323-333 (1996)). There are several ways of testing for gene
transfer into these cells. When gene X encodes a selectable marker
gene, clonogenic 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
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 sorted on a FACS on the basis of their phenotype can be
subjected to PCR analysis specific for the introduced retroviral
vector. The latter can be done on any vector irrespective of the
nature of gene X.
[0086] 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 that any combination of the 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
primates for hematopoietic stem cells can be accomplished by
various methods known in the art.
[0087] 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
[0088] 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 C1 describes a procedure for the 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 hematopoietic 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 the hematopoietic stem cells at
increased gravitational force. Example E1 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 transferof gene X into hematopoietic cells of a
primate according to the invention. Example F1 discloses a
procedure for the establishment of a human stroma cell culture
derived from the natural microenvironment present in the human bone
marrow, and Example F2 describes the use of this stroma cell
culture as a binding compound for the transfer of gene X into
hemopoietic cells of a primate.
Example A
[0089] Production of Selectable Stable Recombinant
Retrovirus-Producing Cells
[0090] In this practical example, use was made of the retroviral
vector construct pLgAL(.DELTA.Mo+PyF101) (Van Beusechem et a!., J.
Exp. Med. 172:729 (1990)), wherein A represents the human cDNA 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., J. Virol. 62:1120 (1988)), according to
the method described by Chen and Okayama (Chen and Okayama, Mol.
Cell. Biol. 7:2745 (1987)). Prior to the transfection, the GP+E-86
cells had been cultured in medium containing 15 .mu.g/ml
hypoxanthine, 250 .mu.g/ml xanthine and 25 .mu.g/ml mycophenolic
acid, so as to select for the preservation of the DNA sequences
responsible for the 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 .mu.M
xylofuranosyl-adenine (Xyl-A) and 10 nM deoxycoformycin (dCF) (Van
Beusechem et al., J. Exp. Med. 172:729 (1990)).
[0091] Then, with the thus obtained cells a ping-pong culture as
described by Kozak and Kabat (Kozak and Kabat, J. Virol. 64:3500
(1990)) was initiated. To that end, 5.times.10.sup.3 transfectants
were mixed with an equal amount of GP+envAm12 amphotropic packaging
cells (Markowitz et al., Virology 167:400 (1988)) and cultured
together in .alpha.-modified DMEM (Dulbecco's Modified Eagle's
Medium) with 10% FCS (Fetal Calf Serum) and 8 .mu.g/ml polybrene.
The amphotropic 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
.mu.g/ml hygromycin B added thereto). The culture was expanded for
two weeks, at which time the amphotropic virus-producing cells were
recovered using the resistance of these cells against hygromycin B.
Individual GP+envAm12 clones that express functional human ADA and
produce the viral proteins, were obtained by culturing limited cell
numbers in medium containing all the above-mentioned components in
the amounts mentioned. In all, 12 of such clones were isolated and
tested.
[0092] 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
murine fibroblasts to dilutions of these supernatants and
subsequently determining the number of fibroblasts that had
acquired resistance against Xyl-A/dCF as a result thereof The
different clones produced between 3.times.10.sup.3 and
2.times.10.sup.5 infective virus particles per milliliter
supernatant. The best clones produced 100.times. more virus than
the best amphotropic LgAL(.DELTA.Mo+PyF101) virus-producing cell
line to date, which had been obtained via a single infection with
ecotropic virus.
[0093] 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 co-cultivated 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
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-resistant
precursor cells could be achieved, while none of the clones showed
a clear toxicity towards these bone marrow cells.
[0094] On the basis of all aforementioned criteria, a cell line was
chosen, which was called POAM-P1. This cell line was 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.
[0095] Two further constructs based on the
pLgXL(.DELTA.Mo.+-.PyF101) 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 construction is described in detail in patent
application WO 96/35798, the contents of which are included herein
by reference. IG-GC-2 contains the full length human placental
glucocerebrosidase (hGC) cDNA, whereas IG-GC-4 has a 160 nt
deletion in the 3' untranslated region of the hGC cDNA. Recombinant
retroviral vector-producing cell lines were generated using the
PA317 cell line with amphotropic host range (Miller and Buttimore,
Mol. Cell. Biol. 6, 2895-2902 (1986)) and using the PG13 cell line
with GaLV host range (Miller et al., J. Virol. 65, 2220-2224(1991))
as described in patent application WO 96/35798. The cell lines were
designated PA2 (PA317 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).
[0096] To harvest batches of recombinant retroviral vector
supernatants, T180 tissue culture flasks were inoculated with
1.times.10.sup.6 virus-producing cells in 25 ml DMDM (Gibco BRL)
supplemented with 10% heat-inactivated fetal bovine serum (FBS) and
the cells were allowed to grow to 90-100% confluency in 4-5 days at
37.degree. C., 10% CO.sub.2 in a 100% humidified atmosphere. Next,
the temperature was changed from 37.degree. C. to 32.degree. C. for
a period of 24 hours before the medium was replaced with 50 ml
fresh culture medium. After an additional culture period of 48
hours, the virus supernatant was harvested, filtered through a 0.45
.mu.m pore size filter, aliquoted and stored at -80.degree. C. The
absence of replication-competent retrovirus (RCR) was tested using
a S.sup.+/L.sup.- foci test after amplification on mus dunni
cells.
[0097] The recombinant retroviral vector titer issuing from the
virus-producing cell lines was established on Gaucher type II
fibroblasts (GM 1260). GMO 1260 cells were seeded at a density of
10.sup.5 cells per 35 mm well (in 6-well plates) in culture medium
further supplemented with polybrene (4 .mu.g/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 14 days as above. Next, genomic DNA
was isolated as described by Stewart et al. (Cell 38 627-637
(1984)). After digestion with NheI and Southern analysis using a
0.65 kb .sup.32p-labeled NcoI-BglI 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 account that the seeded GM01260 cells
probably divided once before the virus supernatant was applied,
approximate virus titers of 3.times.10.sup.5, 1.times.10.sup.5,
2.times.10.sup.5, and 8.times.10.sup.4 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-p1 Described in Example A
[0098] 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 .mu.g/ml DNase I. Cells having a density
lower than 1.064 g/ml were obtained by successively performing a
Ficoll separation and a BSA-density gradient centrifugation (Dicke
et al., Transplantation 8:422 (1969)). 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/liter) .alpha.-modified DMEM, containing 5%
heat-inactivated monkey serum, 15 mg/ml BSA (Bovine Serum Albumin),
1.25.times.10.sup.-5 M Na.sub.2SeO.sub.3, 0.6 mg/ml iron-saturated
human transferrin, 1 .mu.g/ml of each of the following nucleosides:
adenosine, 2'-deoxyadenosine, guanosine, 2'-deoxyguanosine,
cytidine, 2'-deoxycytidine, thymidine and uridine,
1.5.times.10.sup.-5 M linoleic acid, 1.5.times.10.sup.-5 M
cholesterol, 1.times.10.sup.-4 M .beta.-mercaptoethanol, 0.4
.mu.g/ml polybrene, 100 .mu.g/ml streptomycin, 100 U/ml penicillin
and 50 ng/ml of the recombinant rhesus monkey hematopoietic growth
factor IL-3 (Burger et al., Blood 76:2229 (1990)). The thus
obtained cell suspension was seeded at a concentration of
2.times.10.sup.5 cells per cm.sup.2 onto a 70-80% confluent
monocellular layer of POAM-P1 cells, which had shortly before been
exposed to 20 Gray .gamma.-radiation. The bone marrow was
co-cultivated with the POAM-P1 cells for 90 h at 37.degree. C. in a
moisture-saturated atmosphere of 10% CO.sub.2 in air.
[0099] For the duration of the co-cultivation, the rhesus monkey
that had donated the bone marrow was conditioned for the autologous
reception of the co-cultivated 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 co-cultivated 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 trypsinization. A monocellular cell suspension was prepared in
a physiological salt solution with 10 .mu.g/ml DNase I and infused
into a peripheral vein of the donor monkey.
[0100] In order to determine the in vivo regeneration capacity of
the co-cultivated bone marrow, use was made of the
semi-quantitative assay described by Gerritsen et al., (Gerritsen
et al., Transplantation 45:301 (1988)). This method is based on the
observation that the rate at which 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
described by Gerritsen) that the modified bone marrow had preserved
sufficient regenerative capacity and the co-cultivation therefore
had no toxic side effects.
[0101] Analysis at the DNA 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 granulocytes is considered ofgreat 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 ADA 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 introduced human ADA gene in ripe
blood cells could be 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.
Example C
Preclinical Test of a Bone Marrow Gene Therapy Procedure in Rhesus
Monkeys Which Utilizes Purified Hematopoietic Stem Cells
Example C1
Enrichment of Primate Bone Marrow CD34+CD1 lb-Stem Cells
[0102] 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-marke- r CD11b and enriched for cells
carrying the stem cell/precursor cell-marker CD34. This was
performed using immunomagnetic beads, which had been made as
follows: first, tosyl-activated polystyrene magnetic beads
(Dynabeads 450; Dynal, Oslo) were incubated for 24 h in a 0.5 M
borate solution pH 9.5 with 1.25 .mu.g protein A (Pharmacia,
Uppsala) per 10.sup.6 beads. After frequent washing in PBS
containing 0.1% BSA, to the beads, now protein A-coupled,
saturating concentrations of monoclonal antibodies (anti-CD11b:
Mol, Coulter Clone, Hialeah, F1; anti-CD34: ICH3,43) were bound by
incubating for 30 min at room temperature. Finally, the beads were
frequently washed in HBSS/Hepes and stored at 4.degree. C. until
use. The bone marrow cells were incubated for 20 min at 4.degree.
C. with 7 anti-CD11b beads per cell in a concentration of
5.times.10.sup.7 cells/ml at amaximum. Unbound CD11b-negative cells
were stripped from beads and CD 11b-positive cells bound thereto,
using a magnetic particle collector (MPC; Dynal) and washed in
HBSS/Hepes. The thus obtained cells were incubated for 20 min at
4.degree. C. with 5 anti-CD34 beads per cell again in a
concentration of5.times.10.sup.7 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.degree. C. in
HBSS/Hepes with 25% bovine plasma (Gibco, Paisley) and 500 U/ml
heparin.
Example C2
Introduction of the Construct pLgAL(.DELTA.Mo+PyF101) Described
under A) into Rhesus Monkey CD34+CD11b-Stem Cells
[0103] The introduction of the human ADA gene into rhesus monkey
CD34+CD 11b- stem cells and the autologous transplantation
procedure were performed as described under Example b above, the
only difference being that the co-cultivation was performed with
the previously described cell line POC-1 (Van Beusechem eta!., J.
Exp. Med. 172:729 (1990)). As 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.
[0104] After transplantation all blood cell types regenerated
completely, which demonstrates that the gene transfer procedure can
also be performed on CD34+CD11b- stem cells without 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 functional 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 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.
Example D
Introduction of the JG-GC Constructs Described under (A) into Human
CD34+ Hematopoietic Stem Cells by Increased Gravitational Force
[0105] 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 Ficoll gradient
separation according to standard procedures. CD34+ hematopoietic
stem 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.
[0106] 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. The isolated CD34+ cells were seeded at
a cell density of 1.times.10.sup.5 cells/cm.sup.2 in 24-well tissue
culture plates (Greiner) in 400 .mu.l virus supernatant
supplemented with 50 ng/ml interleukin-3 (Sandoz) and 4 .mu.g/ml
protamine sulfate (Novo Nordisk Pharma). 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.degree. C., 10% CO.sub.2 in a 100%
humidified atmosphere. In a 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 ofinfection 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 WO 96/35798) was used that contains
pLgXL(.DELTA.Mo+PyF101) derived recombinant retrovirus vectors
carrying the human multi-drug resistance (MDR]) gene at a titer of
approximately 105 particles per ml as established by vincristine
resistant colony formation of human bone marrow cells.
[0107] 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 with 200 ng/ml SCF, 100 ng/ml IL-6,
100 ng/ml IL-3, 100 U/ml GM-CSF, and 100 ng/ml G-CSF. After a 10
day culture period at 37.degree. C., 10% CO.sub.2 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.degree. C., the clear supernatant was
transferred to new tubes, protein concentrations were measured
(DC-Biorad kit) and lysates were stored at -20.degree. C.
[0108] Glucocerebrosidase activitywas determined with
either4MU-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, 565-574 (1985); Havenga et al., BioTechniques 21, 1004-1007
(1996)).
1TABLE 1 Comparison of 4x supernatant transduction procedure to 4 x
centrifugation enhanced transduction Recombinant Retrovirus
Relative hGC Activity PCR-positive CFU-GM Vector Supernatant
Supernatant Centrifugation Supernatant Centrifugation IGvp010
(negative control) (1) (1) 0/24 (0%) 0/24 (0%) PA2 hGC vector 1 1
1/24 (4%) 5/24 (21%) PG4 hGC vector 1.3 4.5 3/24 (13%) 3/24
(13%)
[0109] Table 1 shows the relative glucocerebrosidase activity data
of this experiment, where the results of cells subjected to
transduction with the JGvp010 retrovirus vector were set at a value
of 1. As can be seen, an increase in hGC activity could not be
detected following transduction with PA2 virus, with or without
centrifugation. In contrast, PG4 virus transduction couldbe
measured by functional hGC activitywhich was 3.5-fold increased
following centrifugation (4.5 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 ml of methylcellulose
medium with cytokines (Methocult GF H4534; Stemcell Technologies,
Inc., Vancouver, Canada) in 6-well plates. After 14 days,
individual colonies were picked and DNA was isolated as described
(van Beusechem et al., Proc. Natl. Acad. Sci. USA 89,
7640-7644(1992)). PCR analysis was performed on this suspension
using oligonucleotide primers 5'-CAGCCCATGTTCTACCAC-3' (SEQ ID NO:
1) and 5'-GGATCCCTAGGCTTTTGC-3' (SEQ ID NO:2). A 50 .mu.l PCR
reaction typically contained 25 pmol of each oligonucleotide, 3%
DMSO, 5 .mu.l 10-times concentrated buffer provided with the
enzyme, 20 pmol dNTP, and 0.25 Units SuperTaq (Promega). The cycler
program consisted of 5 min. 95.degree. C. predenaturation followed
by 26 cycles of each 45 sec. 95.degree. C., 1 min. 54.degree. C., 1
min. 72.degree. C. The program was ended by an elongation step of
10 min. at 72.degree. C. Of the PCR product, 10 .mu.l was run on a
1.5% agarose gel, transferred to a membrane and hybridized with a
0.4 kb .sup.32p-labeled BamHI hGC fragment according to standard
procedures. As can be seen in Table 1, all transductions with hGC
retrovirus vectors yielded PCR-positive colonies, whereas
transductions with the MDR1 control vector did not.
[0110] In experiment 2, PA2, PA4, and PG2 recombinant retroviral
vector supernatants (and IGvpO10 control supernatant) were used to
transduce CD34+cells from normal healthy donor 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.
2TABLE 2 Comparison of a single centrifugation enhanced
transduction procedure to four repeated centrifugation enhanced
transduction procedures PCR-positive CFU-GM/number tested (%)
Recombinant Retrovirus Four rounds of Vector Supernatant Single
transduction 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%)
Example E
Efficient Transfer of Gene X into Human Cd34+ Hematopoietic Stem
Cells Using Recombinant Retroviral Vectors Bound to a Tissue
Culture Dish
Example E1
Production of Peptides Derived From Receptors For Retroviruses
(GLVR)
[0111] The Gibbon ape Leukemia Virus Receptor (GLVR) proteins are
transmembrane molecules expressed on the surface of mammalian
cells. Their primary function is import 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 murine and human mRNA counterparts (Johann, et al,
J. Virol. 66, 1635-1640 (1992); van Zeijl, et al., Proc. Natl.
Acad. Sci. USA 91, 1168-1172 (1994); Weiss and Tailor, Cell 82,
531-533 (1995)). The 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 GLVR1
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 Murine Leukemia
Viruses carrying the 4070A or 10A1 envelope molecules. The GLVR1
homologues from different rodent species have small amino acid
differences in their 4th extracellular domain which determine virus
susceptibility of a cell. Recombinant chimeras between GLVR1 and
GLVR2 proteins suggest that the 4th extracellular domain in GLVR1
is involved in virus binding and infection. Therefore, we have
synthesized peptides encompassing sequences from the 4th
extracellular domain of GLVR1 and GLVR2 using Fmoc chemistry
(performed under contract at Research Genetics, Inc. Huntsville,
Ala., USA). The amino acid sequences of the peptides read from
N-terminus to C-terminus: LVYDTGDVSSKV (SEQ ID NO:3) and
LIYKQGGVTQEA (SEQ ID NO:4) for GLVR1 and GLVR2, respectively.
[0112] For certain applications of the invention, the C-terminus of
thepeptides is extended with 6 Histidine-residues. This enables
coupling to solid support materials via nickle molecules.
Example E2
The Use of GLVR-Peptides as Recombinant Retrovirus Vector Binding
Compounds to Enhance the Transfer of Gene X into Human
Hematopoietic Stem Cells
[0113] 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 .mu.M GLVR1 or 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.degree. C. for 2 hours on GLVR2 or
GLVR1 peptide coated dishes, respectively. This procedure is
repeated twice. Optionally, the thus coated dishes are washed with
PBS with 1% (w/v) human serum albumin (PBS/HAS) and stored at
-80.degree. C.
[0114] Human CD34+ hematopoietic stem cells are obtained as
described under Example D, above, are suspended 1.times.10.sup.6
cells/ml in IMDM (Gibco BRL) supplemented with 50 ng/ml
interleukin-3 (Sandoz), 5% heat-inactivated autologous human serum,
4 .mu.g/ml protamine sulfate (Novo Nordisk Pharma) and 100 U/ml
penicillin (Gist-Brocades) and are cultured for 48 hours at
37.degree. C., 10% CO.sub.2 in a 100% humidified 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.degree. C., 10% CO.sub.2 in a 100% humidified
atmosphere. After this culture, all cells including any adherent
cells are harvested, washed once in PBS/HAS, and used for analysis
of gene transfer or for transplantation by infusion into a
peripheral vein.
Example F
Enhanced Transfer of Gene X into Human CD34+Hemopoietic Stem Cells
Using Recombinant Retrovirus Vectors in the Presence of Human Bone
Marrow Stroma
[0115] Example F1
Establishment of Human Bone Marrow Stroma
[0116] Bone marrow mononuclear cells from healthy donors are
obtained as described in Example D. Five x10.sup.7 cells are seeded
in T75 Nunclon culture flasks (Nunc, Roskilde, Denmark) in 10 ml
DMEM (Gibco) supplemented with 10% heat-inactivated FCS, and
cultured at 37.degree. C., 10% CO.sub.2 in a 100% humidified
atmosphere. Twenty-four hours later the entire medium, including
all nonadherent cells, is removed and replaced with the same medium
further supplemented with 2 mM L-glutamine (Gibco), 10.sup.-4 M
P-mercaptoethanol (Merck, Darmstadt, Germany), and 10.sup.-5 M
hydrocortisone (Sigma) ("stromamedium"). Once aweek, the
stromamedium is replaced with fresh stroma medium. After 3-5 weeks,
a confluent monolayer 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,
respectively. The three lines exhibited similar functional
properties in supporting maintenance of human hemopoietic stem
cells throughout the entire study, i.e., at least during the
culture period ranging from passage 5 to passage 30.
Example F2
The Use of Human Bone Marrow Stroma as a Binding Compound to
Enhance the Retroviral Vector-Mediated Transfer of Gene X into
Human Hemopoietic Stem Cells
[0117] Twenty-four-well tissue culture plates are precoated with
0.3% gelatine (Sigma) in PBS for 16 hours at 4.degree. C. Stroma
cells are seeded 2.times.10.sup.5 cells/well into these plates.
After 1-3 days, confluent stroma monolayers are irradiated with 25
Gy .gamma.-radiation. Immediately thereafter, the irradiated stroma
monolayers are used to support retroviral vector-mediated gene
transfer.
[0118] Serum-free recombinant retrovirus supernatant is harvested
from the cell line IGvp010 (see Patent Application WO 96/35798)
that produces pLgXL(.DELTA.Mo+PyF101) derived recombinant
retroviral vectors carrying the MDR1 gene, either in IGTM
(alpha-modified DMEM containing 1.5% BSA (Sigma), 1 .mu.g/ml of
each of the nucleosides adenosine, 2'-deoxyadenosine, guanosine,
2'-deoxyguanosine, cytidine, 2'-deoxycytidine, thymidine, and
uridine (all Sigma), 1.5.times.10.sup.-7 M Na.sub.2SeO.sub.3
(Sigma), 0.6 mg/ml iron-saturated transferrin (Behring, Marburg,
Germany), 1.5.times.10.sup.-7 M linoleic acid (Sigma),
1.times.10.sup.-4 M 2-mercaptoethanol (Merck, Darmstadt, Germany)
with 5% fetal calf serum (FCS) or in serum-free StemPro-34 SFM
Complete Medium (Gibco RBL Life Technologies, Grand Island, N.Y.).
The retroviral vector supernatant is flash-frozen and stored at
-80.degree. C. until use.
[0119] Human CD34+ bone marrow cells are obtained as described in
Example D. They are suspended 1.times.10.sup.6 cells/ml in IGvp010
supematant in IGTM with 5% FCS or StemPro-34 SFM Complete Medium
supplemented with 50 ng/ml interleukin-3 (Gist-Brocades, Delft, NL)
and 1.6 .mu.g/ml protamine HC1 (Kabi Pharmacia, Woerden, NL) and
are seeded 5.times.10.sup.5 cells per well onto the irradiated
stroma monolayers. Control cultures are started with the same cell
suspensions in culture dishes without stroma monolayers. The cells
are cultured for four days at 37.degree. C., 10% CO.sub.2 in a 100%
humidified atmosphere, and each day the complete medium is replaced
by fresh IGvp010 supernatant and supplements. On day 4, all cells
are harvested by trypsinization as above, and are used for gene
transfer analysis or transplantation.
[0120] For analysis of gene transfer into hemopoietic stem cell and
progenitor cell populations, the cells are stained with a
phycoerythrin-conjugated anti-CD34 MoAb and with a cocktail of
fluorescein isothiocyanate-conjugated Moabs directed against CD38,
CD33, and CD71 as described (Knan-Shanzer et al., Gene Therapy,
3:323-333 (1996)). Populations of CD34.sup.brightCD33
38,71.sup.negative cells, CD34.sup.positveCD33,38,71.sup.positive
cells, CD34.sup.negativeCD33,38,7- 1.sup.positive cells, and
CD.sup.34.sup.negativeCD33,38,71.sup.negative cells are each sorted
separately on a FACStar Plus flow cytometer (Becton Dickinson,
Mountain View, Calif.). Aliquots of 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 added to the sorted
cell samples to reach a total of 10.sup.6 cells per sample. The
cells are pelleted by centrifugation and DNA is isolated from these
cells as described (van Beusechem et al., Proc. Natl. Acad. Sci.
USA, 89:7640-7644 (1992)). The isolated DNA concentration is
measured using PicoGreen DNA Quantitation reagent (Molecular
Probes, Eugene, Oreg.) 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 MDR1 cDNA gene. The sequences of the primers used are:
5'-GTCACCATGGATGAGATTGAG-3' (SEQ ID NO:5) (upstream primer) and
5'-CCACGGACACTCCTACGAG-3' (SEQ ID NO:6) (downstream primer). The
reaction conditions are: 10 mM Tris-HCl pH 9.0, 50 mM KC1, 0.01%
(w/v) gelatin, 0.1% Triton X-100, 1.5 mM MgCl.sub.2 with 200 .mu.M
of all four dNTPs, 200 .mu.M of both primers, and 0.25U SuperTaq
polymerase (HT Biotechnology Ltd. Cambridge, UK) in a total volume
of 50 .mu.l. Forty cycles of 1 minute at 94.degree. C., 1 minute at
55.degree. C. and 1 minute at 72.degree. 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
according to standard procedures (Sambrook et al. Molecular
Cloning. A Laboratory Manual. 2.sup.nd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989)). 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 different phenotype using the data from the FACS reanalysis
of the sorted samples. (See Knan-Shanzer et al. Gene Therapy,
3:323-333 (1996).
[0121] Our invention shows in an example that bone marrow cells
co-cultivated with the virus-producing cells described here are
capable of genetically modifying the hematopoietic system of
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.
[0122] 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 publication or patent application
was specifically and individually indicated to be incorporated by
reference.
[0123] 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.
Sequence CWU 1
1
6 1 18 DNA Artificial Sequence misc_feature Description of
Artificial Sequence primer specific for the IG-GC -2 and IG-GC-4
constructs which contain the full length human placental
glucocerebrosidase (hGC) cDNA and the hGC cDNA with 160 nucleotide
deletion in the 3' region, respectively 1 cagcccatgt tctaccac 18 2
18 DNA Artificial Sequence misc_feature Description of Artificial
Sequence primer specific for the IG-GC-2 and IG-GC-4 constructs
which contain the full length human placental glucocerebrosidase
(hGC) cDNA and the hGC cDNA with 160 nucleotide deletion in the 3'
region, respectively 2 ggatccctag gcttttgc 18 3 12 PRT Artificial
Sequence misc_feature Description of Artificial Sequence peptide
based on the 4th extracellular domain of Gibbon ape Leukemia Virus
Receptor protein 1. 3 Leu Val Tyr Asp Thr Gly Asp Val Ser Ser Lys
Val 1 5 10 4 12 PRT Artificial Sequence misc_feature Description of
Artificial Sequence peptide based on the 4th extracellular domain
of Gibbon ape Leukemia Virus Receptor protein 2. 4 Leu Ile Tyr Lys
Gln Gly Gly Val Thr Gln Glu Ala 1 5 10 5 21 DNA Artificial Sequence
misc_feature Description of Artificial Sequence primer specific for
the human multi drug resistance gene 1 (MDR1) cDNA 5 gtcaccatgg
atgagattga g 21 6 19 DNA Artificial Sequence misc_feature
Description of Artificial Sequence primer specific for the human
multi drug resistance gene 1 (MDR1) cDNA 6 ccacggacac tcctacgag
19
* * * * *