Note: Descriptions are shown in the official language in which they were submitted.
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PROMOTION OF SELF-RENEWAL AND IMPROVED GENE
TRANSDUCTION OF HEMATOPOIETIC STEM CELLS
BY HISTONE DEACETYLASE INHIBITORS
This invention relates to the field of self renewal divisions and transduction
of stem
cells, particularly hematopoietic stem cells (HSCs).
The maintenance of the hematopoietic system relies on primitive pluripotent
hematopoietic stem cells that have the capacity to self renew and repopulate
all the blood cell
t.o lineages with relevant progenitor cells. Due to their capacity for self
renewal and their
pluripotent, long term reconstituting potential, HSCs have long been
considered ideal for
transplantation to reconstitute the hematopoietic system after treatment for
various
hematologic disorders or as a target for the delivery of therapeutic genes.
Additionally, human
HSCs have potential applications in restoring the immune system in autoimmune
diseases and
;t5 in the induction of tolerance for alogenic solid organ transplantation.
Early in hematopoiesis, a pluripotent stem cell differentiates and gives rise
to either
lymphoid, myeloid, or erythroid restricted cells. These cells in turn
differentiate into
progenitor cells that are committed to a specific cell type. Evidence
indicates these progenitor
?0 cells have lost the capacity for self-renewal and contribute little if
anything to engraftment
following transplantation. It appears that infused HSCs are necessary for
short term and
sustained engraftment. Furthermore, the kinetics of engraftment is
proportional to the number
of infused HSCs. This is particularly true in allotransplantation settings.
Therefore attempts to
improve graft quality may be accomplished if there is an increase in HSC
number.
:zs Additionally, increase in HSC number by culturing in vitro will allow
improved therapeutic
approaches for the treatment of many diseases including cancer, autoimmune and
certain
genetic diseases.
HSCs are generally obtained from bone marrow, mobilized peripheral blood and
3o umbilical cord blood. Presently large volumes of bone marrow must be
extracted and frozen
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to ensure that sufficient number of stem cells are present to reconstitute the
hematopoietic
system. Additionally, single cord blood donors provide insufficient numbers of
hematopoietic
stem cells for adult therapeutic use. Therefore, methods developed for
promotion of HSC
self renewal as distinguished from stem cell expansion could provide a
tremendous advantage
for therapeutic uses.
Since the 1980's, numerous discoveries have been made concerning the influence
of
growth factors, such as interleukins and cytokines, on hematopoietic cells.
These hormones
exert a profound effect on the growth and differentiation of hematopoietic
cells. Some of
these molecules include interleukin (IL) -1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-
7, erythropoietin
(EPO), granulocyte-colony stimulating factor (G-CSF), macrophage-colony
stimulating factor
(M-CSF), and granulocyte-macrophage-colony stimulating factor (GM-CSF). In an
attempt to
achieve optimal production of progenitors during in vitro culture, these
factors have been
tested in many combinations. A number of major breakthroughs in stem cell
replication came
with the discovery of c-kit ligand and a ligand for the fetal liver kinase
receptor, fik2/flk3
ligand (FL). Both of these cytokines synergize with other cytokines in vitro
and have the
effect of sustaining viability and recruiting very primitive hematopoietic
cells into cycle
without increasing their differentiation. Additionally, the molecule
thrombopoietin (TPO)
which promotes megakaryocytopoiesis was discovered to have a potent effect on
the viability
2o and replication of both human and murine primitive hematopoietic progenitor
cells in vitro.
Other culture conditions and additives have been studied with the hope of
optimizing HSC
replication. These aspects of t:he culture system include the culture media,
the surface for
adhesion, a.nd schedules for replacement of gases and nutrients. The role of
stroma cells has
also been investigated in stem cell self renewal technology. Cell adhesion
molecules expressed
on the surface of primitive cells, such as VLA-4, VLA-5 and L-selectin have
been explored
for effects on stem cell cycling. Specific receptor-ligand interactions
between stromal cells and
HSCs have been identified that appear to be critical for stem cell
maintenance.
While the manipulation of culture conditions is one approach to promote HSC
self
3o renewal, the present invention is concerned with the transcriptional
regulation of genes
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involved in self renewal through l:he modulation of histone acetylation.
In eukaryotic cells, DNA molecules are tightly complexed with histone proteins
to
form chromatin, and changes :in chromatin structure are known to be of
fundamental
importance in regulation of gene. expression. Histones are rich in arginine
and/or lysine, and
are classified in five major groups designated H1(lysine-rich), H2A and H2B
(slightly lysine
rich), and H3 and H4 (arginine rich). These proteins have two domains, the
histone fold
domain and the amino tail domain. The amino tail domain interacts with DNA at
specific sites
between the positively charged arginine/lysine residues and the negatively
charged phosphate
groups of the DNA. At specific: points in the cell cycle, the amino tail
domain undergoes
transient modifications including acetylation. The histone-modifying enzymes,
particularly
acetyltransferases and deacetylases, are known to play active roles in the
transcription and
assembly of chromatin. While not meant to be limiting, the prevailing
scientific view is that a
correlation exists between the level of histone acetylation and deacetylation
and
't5 transcriptional activity, and that histone acetylation leads to a
decondensation of chromatin
which facilitates the access of transcriptional factors and components of the
basal
transcriptional machinery to DNA. (Grunstein, Michael, Nature 389:349 - 352,
{1997); Wade,
P. et al., TIBS 22:128 - 132, (1997)). It is also believed that some
transcriptional cofactors
(activators) have histone acetyl transferase activity, and therefore these
coactivators could
z0 direct local destabilization of repressive histone-DNA interactions.
Conversely, a growing number of transcriptional repressors have been found to
act
through the recruitment of histone deacetylases (HDACs). Several repressors of
transcription
are involved in the mechanisms that govern cellular differentiation and
proliferation and have
25 been implicated in normal hematopoiesis or leukemogenesis. Some of these
repressors are
briefly mentioned.
Retinoid receptors are involved in myeloid maturation but may also play a
critical role
30 in the development of pluripotent hematopoietic stem cells (Tsai, et al.
Genes & Dev. 8: 2831
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(1994)). While these receptors l~~have as transcriptional activators when
bound to their ligand,
in the non-ligand state they repress the basal transcriptional activity by
associating with the
transcriptional corepressors SMRT and N-CoR. Nagy, et al. has shown that the
SMRT
transcriptional corepressor exerts its activity by forming a multisubunit
repressor complex in
which the histone deacetylase 1 (HDAC1) is recruited. (Nagy, et al. Cell, 89:
373 - 380
(1997)) It was confirmed that lnistone deacetylation is fundamental for
retinoid signaling by
showing that the deacetylase inhibitor trichostatin A (TSA) could greatly
enhance the
biological activity of retinoic acid. This was demonstrated using the HL60
promyelocytic cell
line that undergoes myeloid maturation in response to retinoic acid. In the
presence of sub-
optimal doses of retinoic acid, a full differentiative response of the cells
was achieved by
simultaneous treatment with TS.A.
Promyeiocyctic leukemia zinc finger (PLZF) protein is predominantly expressed
in the
most primitive hematopoietic compartment and has been shown to inhibit cell
cycle of
hematopoietic cells (Mol. Cell Biol.18: 5533( 1998)). PLZF is a DNA binding
protein that
represses transcription by interacting with either SMRT or N-CoR which recruit
histone
deacetylases (Guidez, et al. Blood 91: 2634 {1998)) and Lin, et al. Nature
391: 811 (1998)).
Activation of myc by genomic alterations resulting from chromosomal
rearrangements
or retroviral insertion occurs iti human, rodent and avian leukemias and
lymphomas. MYC
proteins are expressed in proliferating cells and are down regulated upon cell-
cycle withdrawal
or differentiation. MYC is part of a transcriptional network that involves
other factors. MYC
can dimerize with a protein called MAX which can bind to DNA and to the
protein MAD.
MYC-MAX dimers are transcriptionally active, whereas MAD-MAX dimers repress
transcription. It has been shown that MAD-MAX recruits histone deacetylases
and that this
interaction is the basis for the IV4AX-MAD repressive activity. TSA has been
shown to abolish
the repression. (Laherty, et aL t:ell 89:349 - 356 (1997)).
Another co-repressor whose activity can be modulated by histone deacetylase
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inhibitors is the mammalian CBFI/RBP-Jx which switches from a transcriptional
repressor to
an activator upon Notch activation. In the absence of Notch activation
CBF1/RBP-Jx
represses transcription by bindvig to SMRT/HDACI, while Notch activation
disrupts the
formation of the repressor complex. TSA treatment can induce the transcription
of CBFl
regulated genes. (Genes and Dev., I2: 2269-2277 ( 1998)). Notch signaling has
been shown to
influence the development of primary primitive hematopoietic precursor cells
in vitro (Blood
1998, 91: 4084-4091) and overexpression of the activated Notchl in the 32D
myeloid cell line
inhibits differentiation and perrruts expansion of undifferentiated cells,
findings consistent with
the known function of Notch in Drosophila (PNAS 1996, 93:13014-9).
In a recent review article, it was disclosed that the HDAC inhibitor,
trichostatin A
(TSA) induced a variety of biological responses in cells including induction
of differentiation
and cell cycle arrest. (Yoshid.a, et al., BioEssays 17:42-429( 1995)).
Additionally various
inhibitors of HDAC have been implicated in triggering terminal differentiation
of malignant
cells. (Richon, et al., Proc. Natl. Acad. Aca. USA, 95:3003-3007 (1998)).
While the literature di.~;closes the use of HDAC inhibitors in studies to
reverse the
effects of HDAC and HDAC complexes in the repression of transcription, the
literature does
not disclose a method of using HDAC inhibitors to promote hematopoietic stem
cell cycling,
to promote HSC self renewal divisions (or replication), nor to enhance
maintenance of
primitive cell function during gene integration over gene integration in the
absence of histone
deacetylase inhibitors.
In one aspect of the invention, a method of promoting self renewal division of
hematopoietic stem cells is provided, comprising obtaining a population of
hematopoietic cells
from a source of hematopoietic cells, wherein the obtained hematopoietic cells
include a
subpopulation of hematopoietic stem cells; culturing the hematopoietic cells
under growth
supporting conditions; exposiing the cultured cells to an effective amount of
a histone
deacetylase inhibitor wherein self-renewal divisions of the stem cells is
promoted; and
obtaining a composition of thc: self-renewed hematopoietic stem cells. The
method may also
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include a further step comprising obtaining a population of enriched
hematopoietic stem cells
from the hematopoietic cells prior to culturing the stem cells. Preferably the
method includes
selection of enriched human hematopoietic stem cells from the phenotype group
consisting of
CD34+; Thy-1+; CD34'Thy-I+; CD34-Thy-1+; CD34+EM''; CD3f'Thy-1+Liri ;
CD3f"CD38'°'-
; CD34'Thy-1+CD381°~- ; CD34'Thy-1~'EM+; and CD3f'Thy-1+CD381°~-
EM+. In one
embodiment, the invention includes the composition of self renewed cells
produced by the
method defined above.
1. In another aspect of the invention, a culture is provided, comprising
isolated
1o mammalian hematopoifaic cells which include a subpopulation of engrafting
cells
capable of self renewal, an effective amount of one or more histone
deacetylase
inhibitors, and effective amounts of growth and self renewal supporting
cytokines
wherein the effective amount of the histone deacetylase inhibitor promotes
self
renewal division of the engrafting cells in the culture. In one preferred
embodiment,
the hematopoietic cells are human. In another prefered embodiment, the histone
deacetylase inhibitor is selected from the group trichostatin A, trapoxin,
chlamydocin,
sodium butyrate or dimethyl sulfoxide.
In a further aspect of the invention, a method of promoting self renewal
division of
2o stem cells is provided, comprising treating a population of stem cells with
an effective amount
of one or more histone deacetylase inhibitors and allowing the stem cells to
self renewal. In
one embodiment the stem cells are enriched hematopoietic stem cells
characterized as
CD34'Thy-1+.
In an additional aspect, the invention provides a method of generating
transduced
mammalian stem cells comprising treating stem cells in a culture with an
effective amount of a
histone deacetyiase inhibitor, :introducing a gene into the cultured stem
cells using retroviral
mediated transfer, and allowing transduction of the stem cells wherein the
number of
transduced stem cells is increased over the number of transduced stem cells
exposed to
3o substantially the same conditions but in the absence of treatment with the
histone deacetylase
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inhibitor. In a preferred aspect, the stem cells are hematopoietic stem cells,
preferably human
hematopoietic cells, and the gene is a therapeutic gene. The method is further
comprised of
administering an effective amount of a population of the transduced stem cells
to a mammalian
subject, preferably, a human subject. In one embodiment the stem cells are
allogeneic or
xenogeneic to the subject anti in another embodiment the stem cells are
autologous to the
subject.
In yet a further aspect, the invention provides a method of genetically
modifying stem
cells by contacting a gene delivery vehicle comprising a polynucleotide with a
population of
l0 stem cells cultured in the presence of an effective amount of a histone
deacetylase inhibitor,
and obtaining genetically modified stem cells. In a preferred embodiment, the
delivery vehicle
is a retroviral vector, a lentivira vector, an adenoviral vector or a liposome
delivery vehicle.
In yet a further aspect., the invention provides a method of restoring
hematopoietic
capability in a subject, comprising contacting a population of hematopoietic
stem cells with an
effective amount of a histone deacetylase inhibitor; transducing the
hematopoietic stem cells
by exposing the hematopoietic stem cells to a vector including a nucleic acid
sequence
encoding a therapeutic gene; and administering an effective amount of a
population of the
transuded hematopoietic stem cells to a subject wherein hematopoietic
capability is restored.
In another aspect, the invention provides a method of improving engraftment of
genetically modified mammalian stem cells comprising exposing stem cells in a
culture to an
effective amount of a histone deacetylase inhibitor, introducing a
heterologous gene into the
cultured stem cells, generating an increase in the number of genetically
modified stem cells
over that in the absence of exposure to a histone deacetylase inhibitor, and
administering the
modified cells to a subject. In one embodiment, the stem cells are allogeneic
and in another
embodiment, the stem cells are autogolous. Preferably the heterologous gene is
a therapeutic
gene.
A further aspect of the invention provides a method of transducing stem cells
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comprising exposing a population of stem cells to an effective amount of a
histone deacetylase
inhibitor; transducing the stern cells with a foreign gene; and obtaining
transduced cells
wherein the number of transduced stem cells is increased over the number of
transduced stem
cells grown under substantially the same conditions but in the absence of
exposure to an
effective amount of a histone deacetylase inhibitor. In one embodiment the
cells are
transduced in culture and in a second embodiment the cells are transduced in
vivo. In a further
embodiment the stem cells arc: hematopoietic stem cells, particularly mice or
human. In yet
another embodiment, the cells are transduced with a retroviral vector derived
from Moloney
murine leukemia virus or murvae stem cell virus.
l0
In yet an additional aspect, the invention provides a culture comprising a
population of
stem cells; an effective amount of one or more histone deacetylase inhibitors;
an effective
amount of one or more growth supporting cytokines; and a gene delivery vehicle
including a
polynucleotide encoding a marker gene or therapeutic gene. In one embodiment
the gene
delivery vechicle is a vector derived from a retrovirus, adenovirus or adeno-
associated virus.
Figure 1 illustrates the increased number of human hematopoiefic progenitor
cells
expressing the Thy-1 antigen from a culture of MPB CD34+ cells exposed to HDAC-
inhibitors.
25
Figures 2A and 2B illustrate that a culture of human MPB CD34+ cells exposed
to the
HDAC inhibitor chlamydocin increases total CAFC activity within the culture
relative to
cultures without chlamydocvi. Figure ZA shows the total CAFC activity among
Thy-1+ cells
and Figure 2B shows the tota CAFC activity among cells.
Figure 3 illustrates that a larger proportion of human CD34+ hematopoietic
progenitor
cells cultured with chlamydocin retain expression of the Thy-1 antigen
throughout 4 divisions
in culture (bottom) than cells cultured without HDAC-inhibitors (top).
Figure 4 illustrates that the proportion of Thy-1+ cells expressing the
myeloid lineage
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antigen CD 15 is not upregulat~ed on human CD34+ MPB cells cultured with HDAC-
inhibitors.
Figures SA, 5B and 5C: illustrate the profile of Lin, Thy-1, c-kit, and Sca-1
expression
among viable murine HSCs analyzed four days after growth in media alone (A) or
media
supported with TSA (B) or chlamydocin (C).
Figure 6 illustrates engraftment in NOD SCID marrow of MPB CD34+ cells
cultured
in TPO, FL, and KL with 24 hour incubation with Chlamydocin.
1o The following abbreviations used through out the disclosure are listed
herein below:
FITC = fluorescein
TPO = thrombopoietiti
FL = Flt3 ligand
KL = c-kit ligand
IL = Interleukin
LIF = leukemia inhibitory factor
MPB = mobilized peripheral blood
CFSE = carboxyfluorescein-diacetate succinimidylester
HSC = hematopoietic stem cell
TSA = Trichostatin A
DMSO = dimethyl sulfoxide
FBS = fetal bovine serum
IMDM = Iscove's modified Dulbecco's medium
HDAC = histone deacetylase
HDAC-I = histone deacetylase inhibitor
CAFC = cobblestone-area-forming cell
PE = phycoerythrin
CM = culture medium
3o APC = allophycocyansn
EPO = erythropoietin
FN = fibronectin fragment CH296
FACS = fluorescence-activated cell sorter
As used herein the term "stem cell" includes stem cells of various cell types,
such as,
muscle, epithelial, neural and bone stem cells. More particularly the
invention is concerned
with hematopoietic stem cells. The term "hematopoietic stem cell" (HSC) refers
to
mammalian and avian hematopoietic stem cells and means a population of
hematopoietic cells
containing the engrafting potential for in vivo therapeutic application.
Hematopoietic cells
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encompass not only I3SCs, but ;also erythrocytes, neutrophils, monocytes,
platelets, mast cells,
eosinophils and basophils, B and T lymphocytes and NK cells as well as the
respective lineage
progenitor cells. Stem cells ma.y also be defined in vitro by the presence of
CAFC activity.
Animal models for long term engrafting potential of candidate human
hematopoietic stem cell
populations include the SCID-hu bone model (Kyoizumu, et al., Blood 79:1704
(1992); and
Murray et al., Blood 85: 368 - 378 (1995) ), the in utero sheep model
(Zanjani, et al., J. Clin.
Invest. 89:1179 ( 1992)), NOD SCID (Larochelle, et al., Nature Medicine 2:
1329 - 1337
(1996) and Conneally, et af, Proc. Natl. Acad. Sci. 94:9836 - 9841 (1997)) and
primate
models (Kiem, et al., Blood 90:4630- 4645 (1997) and Srour, et al., J.
Hematother 1:143 -
153 (1992)). A mouse hematopoietic stem cell has been obtained in at least
highly
concentrated form where fewer than about 30 cells obtained from bone marrow
were able to
reconstitute all of the lineages of the hematopoietic system of a lethally
irradiated mouse. Each
assayed cell was multipotent for all hematopoietic lineages.
An engrafting cell mews any cell that can lodge in a hematopoietic tissue and
function
to repopulate blood cell linea,ges. Additionally, as used in the specification
and claims, the
singular form "a", "an", and "l:he" include plural references unless the
context clearly dictates
otherwise. For example, a stem cell includes a plurality of cells. The term
"self renewal" or
"self renewal divisions" also referred to as "replication", is defined herein
to mean cell division
without apparent cell differentiation or without loss of cell engrafting
capacity. The term
"expansion" is intended to mean allowance of progenitor cells to increase in
number and
differentiate from the pluripotE:nt stem cells used to initiate the culture.
As used herein the term "cytokine" refers to any one of the numerous factors
that
exert a variety of effects on cells, for example, inducing growth or
proliferation. The cytokines
may be human in origin, or may be derived from other species when active on
the cells of
interest. Included within the scope of the definition are molecules having
similar biological
activity to wild type or purified cytokines, for example produced by
recombinant means; and
molecules which bind to a cytolcine factor receptor and which elicit a similar
cellular response
as the native cytokine factor.
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Non-limiting examples of cytokines which may be used alone or in combination
in the
practice of the invention include, but are not limited to, IL-2, IL-3, IL-6,
IL-12, IL-la, IL-11,
KL, (designated interchangeably with c-kit ligand, stem cell factor (SCF),
steel factor (Stl)
and mast cell growth factor (MCGF)), granulocyte colony stimulating factor (G-
CSF),
granulocyte-macrophage colony stimulating factor (GM-CSF), TPO, LIF, FL, and
MIP-la.
As combinations, fused proteins may be employed, where the two factors are
fused together
or a cytokine is fused to its soluble receptor. The order of fusion will not
be critical so long as
the two portions of the molecules act independently and provide for their
biological function.
l0 A non-limiting example includes combinations of IL-3 and IL-6 (Broxmeyer,
et al, Exp.
Hematol. 18:615 ( 1990). The present invention also includes culture
conditions in which one
or more cytokines is specifically excluded from the medium. Cytokines are
commercially
available from many vendors such ass R & D Systems Inc., (Minneapolis, MN),
Genzyme
Diagnostics (Cambridge, MA) and Genentech (South San Francisco, CA).
The term "culturing" refers to the propagation of cells or organisms on or in
media of
various kinds.
An "effective amount" of a cytokine is an amount sufficient to promote
survival,
24 growth, and/or division of stem cells, particularly hematopoietic stem
cells. "Survival" is
defined herein as the ability to continue to remain alive or function.
As used herein the term "mammal" includes but is not limited to humans, mice,
monkeys, farm animals, sport animals, pets, and other laboratory rodents and
animals.
Preferably the term refers to humalns.
"Histone deacetylase activity" or " the activity of a histone deacetylause
protein" refers
to the biochemical activity associated with histone deacetylase proteins. The
biochemical
activity includes binding to and optionally catalyzing the deacetylation of am
acetylated
histone; this may result in mediating the activity of transcriptional co-
repressors such as, but
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not limited to, SMRT and N-CoR. In general, compounds that inhibit HDAC have
been
shown to result in activation of gene expression.
An effective dose or amount of a histone deacetylase inhibitor is defined as
the amount
of a HDAC-I which increases the number of self renewal divisions of stem cells
within a
population of cells, particularly hematopoietic cells wherein overall cell
viability may or may
not be reduced and as a result increases the number of stem cells available
for genetic
modification as compared to stem cells within a popluation of cells grown
under essentially
the same conditions but in the .absence of HDAC-I. .
As used herein, the term "genetic modification" refers to any addition,
deletion or
disruption to a cell's normal nucleotides. The method of this invention is
intended to
encompass any genetic modification method of exogenous or foreign gene
transfer (or nucleic
acid sequence transfer) into stem cells (preferably into hematopoietic stem
cells). The term
~5 "genetic modification" encompasses use of a gene delivery vehicle and
includes but is not
limited to transduction (viral mediated transfer of nucleic acid to a
recipient, either in vivo or
in vitro), transfection (uptake by cells of isolated nucleic acid), liposome
mediated transfer
and others means well known in the art.
20 A "therapeutic gene" is defined herein as an entire gene or only the
functionally active
fragment of the gene. The therapeutic gene may be capable of compensating for
a deficiency
in a patient that arises from a defective or absent endogenous gene.
Additionally, a
therapeutic gene may be one that antagonizes production or function of an
infectious agent,
antagonizes pathological procEases, improves a host's genetic makeup,
facilitates engraftment,
25 or a stem cell's therapeutic potency.
As used herein, the term "retroviral mediated gene transfer" and "retroviral
transductian" are used interchangeably.
3o Methods of obtaining hematopoietic cells and stem cells are well known in
the art and
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include isolation of the stem cells from other cells in hematopoietic tissues
of the body and
particularly bone marrow. Ste;m cells from bone marrow appear to be in the
range of about
0.01 to about 0.1 % of the bone marrow cells. Bone marrow cells may be
obtained from ilim,
sternum, tibiae, femora, spine or other bone cavities. Other non-limiting
sources of
s hematopoietic stem cells include embryonic yolk sac, fetal liver, fetal and
adult spleen, blood,
including adult peripheral blood and umbilical cord blood. (To, et al., Blood
89:2233-2258
(1997)).
For the isolation of bane marrow an appropriate solution may be used to flush
the
bone, including but not limited to, salt solution, supplemented with fetal
calf serum or other
naturally occurring factors in conjunction with an acceptable buffer at low
concentrations,
generally about 5 to 25 mM. Buffers include but are not limited to HEPES,
phosphate and
lactate buffers. Bone marrow can also be aspirated from the bone in accordance
with
conventional techniques.
1s
The manner in which stem cells may be separated or selected from other cells
is not
critical to this invention. Various procedures which may be employed include
magnetic
separation using antibody coated magnetic beads, affinity chromatography, and
cytotoxic
agents joined to a monoclonal antibody. Also included is the use of
fluorescence activated cell
sorters (FACS) wherein the ce;lls can be separated on the basis of the level
of staining of the
particular antigens. These techniques are well known to those skilled in the
art and reference
is made to U.S. Patent Nos. 5,()61,620; 5,409,8213; 5,677,136; and 5,750,397;
and Yau, et
al., ( 1990) Exp. Hematol. 18:219-222.
The order of separation is not critical to this invention. However, preferably
cells are
initially separated by a coarse separation followed by separation using
positive and/or negative
selection. In such positive selE:ctions, up to about 15%, usually not more
than about 10%,
preferably not more than about 5% and most preferably not more than about 1%
of the total
cells in the retained cell population will lack the marker used for
separation. In some
instances it may be desirable to directly treat a hematopoietic cell
population with an effective
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dose of a histone deacetylase inhibitor without first separating a
subpopulation of HSCs from
the hematopoietic cells.
Stem cells, which constitute only a small percentage of the total number of
hematopoietic cells are characterized by both the presence of markers
associated with specific
epitopic sites identified by antibodies (positive selection) and the absence
of certain markers as
identified by the lack of bindnng of specific antibodies. All of the
monoclonal antibodies
(mAbs) that react with a particular membrane molecule are grouped together as
a cluster
designator (CD). Lin~ cells refer to a cell population selected on the basis
of the lack of
1o expression of at least one lineage specific marker. For example, those
human cells which lack
markers associated with T cells (such as CD2, CD3, CD4, and CD8), B cells
(such as CD10,
CD 19 and CD20), myeloid cells; (such as CD 14, CD 15 and CD33), natural
killer cells (such
as CD2, and CD56), and the like:.
Human stem cells may be characterized by the following non-limiting
phenotypes:
CD2 , CD3 , CD4 , CD7 , CD8 , CD 10 , CD 14 , CD 15 , CD 19 , CD20 , CD33 ,
CD34 ,
CD34+, CD38j°/ , CD45RA , C:D59+/ , CD71 , CDW109+, glycophorin , Thy-
1''(CD90),
HLA-DR+/ , AC133+, rhodamine 123 (rho123~°) or a combination thereof.
Monoclonal
antibodies to the molecule Thy-1 in combination with mAbs to CD34 have been
used to
isolate murine, non-human primate and human HSCs. USP 4,714,680 describes a
population
of cells expressing the CD34 marker. More recently other mAbs have been
identified for
positive selection of human stern cells. These are designated EM,
specifically, EMS, EM10
and EM16 (Chen, et al., Immunological Reviews, 157: 41 - 51, 1997). As used
herein EM+
means an EM phenotype that expresses any EM marker.
:!5
In one preferred embodiment the surface antigen expression profile of an
enriched
hematopoietic stem cell population will be selected from the following group,
CD34+; Thy-1'';
CD10-; CD19-; CD15~; CD33-; CD34''Thy-1+; CD34 Thy-1+; CD34''EM+;
CD34+CD381°~-;
CD34''Rhol2s; CD33 Thy-1+; CDl9~Thy-1+; CD34''Thy-1'Liri ; CD34''Thy-1'EM+;
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CD34+CD45RA CD71 ; HLA-DR+/-CD34'' Thy-1+I,iri ; CD34iThy-
1+CD381°~'; and
CD34;'Thy-1+CD381°~~EM+. In .a more preferred embodiment the expression
profile of an
enriched population will be selected from CD34''; Thy-1+; CD34~Thy-1+; CD34
Thy-1+;
CD34~EM+; CD34+CD381°/-; C'D34'Thy-1+Liri ; CD34'"Thy-1+EM+; CD34''Thy-
1+CD381°~-;
and CD34+'Thy-1+CD381°~-EM+, particularly when EM+ is EM10+. The
combination of
markers used to isolate and define an enriched HSC population may vary as
other markers
become available.
Generally the number of cells obtained after positive selection will be fewer
than about
20% of the original cells, more frequently fewer than about 10%, generally
fewer than 5%,
most frequently fewer than 1.0 io, and may be as low as 0.2% or less.
Compositions having
greater than 80%, usually greater than about 90% of human stem cells may be
achieved by the
enumerated separation techniques when the desired stem cells are identified by
being
CD34i"Thy-1 ~''L,iri .
is
Murine (m)HSCs are included in the Lin°°gn° population
which corresponds to cells
that express at the most dim levels of a lineage specific marker. For example
those cells which
lack expression of, or express love levels of T lymphocytes markers (such as
CD2, CD3, CD4,
CDS
20 or CD8), B lymphocytes markers (such as CD19 or B220), myeloid cells
markers (such as Gr-
1 or Mac-1=CDllb), natural killer markers (such as NK1.1) or erythroid markers
(such as
Ter119).
Murine HSCs may be ch~uacterized by the following non-limiting phenotypes: CD2-
;
2.5 CD3-; CD4' ; CD4'°'"; CDS- ; CD8- ; CD19- ; B220-; Gr-1-; Mac-1-;
Mac-1'°; NK1.1-;
Ter119~; Ter119'°; Sca-1+;Thyl.l''; c-Kit - or c-Kit~"~"; CD34-; CD34+;
CD38 ~'~'; CD43~'~';
H-2K ~'~'; or AA4.1+. The combined use of monoclonal antibodies and
fluorescent vital dyes
such as the mitochondria-binding dye Rhodamine 123 (Rho-123) or the DNA-
binding dye
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Hoechst 33342 can increase the enrichment of HSC in a sorted population. The
most primitive
cells are enriched in the Rho-12:3'° fraction as well as in the Hoechst
33342'° fraction.
In a preferred embodiment, an enriched murine HSC population is characterized
by
Thy-1'°; Liri "°; Sca-1+; and c-lit+. In a more preferred
embodiment the enriched population
is sorted by or selected from the phenotypes Thy-1'°""Lin"° Sca-
1+; c-Kit'Lin"°Sca-1+; c-
Kit+Sca-1+or c-Kit+Thy-llowL~..n°Sca-1+.
In mice HSC can be largely separated, in vitro or in vivo, from more mature
to progenitors by their differential sensitivity to cytotoxic drugs such as 5-
fluoruracil. Further
separation of murine HSC can be achieved using counterflow centrifugal
elutriation (CCE) on
the basis of cell size and density; the small cell subset obtained by CCE at a
flow rate of
25mL./min contains HSC with long term repopulating activity and very few
progenitors
(Jones, et al. Nature 347:188 ( 1990)).
:l5
Once hematopoietic cells, are harvested, and HSCs optionally separated, the
cells are
cultured in a suitable medium under growth supportive conditions which
includes a
combination of growth factors that are sufficient to maintain the growth of
hematopoietic
cells. Any suitable container, flack, chamber, bag, bioreactor with perfusion
or appropriate
2 0 vessel such as a 24 well plate or the like can be used. Culture containers
are readily available
from commercial vendors. While the seeding level is not critical and will
depend on the type of
cells used, in general the seeding level will be at least about 10 cells per
ml, more usually at
least about 100 cells per ml and generally not more than about 2 x 106 cells
per ml and usually
not more than 105 cells per ml when the cells are CD34+.
Various culture media ca.n be used and non-limiting examples are Iscove's
modified
Dulbecco medium (IMDM), :K-vivo 15 (serum free), and RPMI-1640. These are
commercially available from various vendors for example JRH BioSciences. The
formulations
can be supplemented with a variety of different nutrients, growth factors,
cytokines and the
3~D like. The medium can be serum free or supplemented with suitable amounts
of serum such as
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fetal calf serum or autologous serum or plasma. Preferably, if the cells or
cellular products are
to be used in humans, the medium will be serum free or supplemented with
autologous serum
or plasma. In general, the medium will include effective amounts of cytokines,
particularly
TPO, KL, FL and ILs. One skniled in the art is aware of the various growth
supporting culture
media and culturing techniques for HSC's and reference is made to Lansdorp, et
al., J. Exp.
Med. 175:1501 (1992) and Petzer, et al., PNAS 93:1470 (1996).
In one aspect the medium formulation is supplemented with TPO. A preferred
concentration range is from albout 0.1 ng/mL to about 500 p.g/mL, more
preferred is from
about 1.0 ng/mL to about 1000 ng/mL, even more preferred is from about 5.0
ng/mL to about
300 ng/mL, and most preferred is from about 10.0 ng/mL to about 100 ng/mL.
Additionally,
the medium will include Flt3 ligand (FL) and c-kit ligand (KL) each
individually at a preferred
concentration range from about 0.1 ng/mL to about 1000 ng/mL, more preferred
from about
1.0 ng/mL to about 500 ng/ml,, and even more preferred from about 10 ng/mL to
about 300
is ng/mL.
While various interleukins may be used to supplement the medium, IL-6 is
preferred.
A preferred concentration range is from about 0.1 ng/mL to about 500 ng/mL,
more preferred
from about 1.0 ng/mL to about 100 ng/mL, and most preferred from about 5 nglmL
to about
2o SO ng/mL. Hyper IL-6, a high ;affinity or covalent complex of IL-6 and
soluble IL-6 receptor,
may also be used and reference is made to Conneally, et al., Ann. Meeting of
the International
Soc. for Exper. Hematology, Vancouver, Abst. #60 (1998).
Other cytokines may be added individually or in combination and include but
are not
25 limited to IL-1, IL-2, IL-3, Il.-6, IL-12, IL-11, stem cell factor, G-CSF,
GM-CSF, Stl ,
MCGF, LIF and MIP-la. When murine stems cells are cultured, a preferred non-
limiting
medium includes mIL-3, mIL-6 and mSCF. A preferred concentration range is from
about O.I
ng/mL to about 1000 ng/mL, more preferred is from about 1.0 ng/mL to about 500
ng/mL,
and most preferred is about 5.0 ng/mL to about 200 ng/mL.
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In one embodiment, thc: cytokines are added to the media and replenished by
media
perfusion. Alternatively the cytokines may be added separately, without media
perfusion, as a
concentrated solution through separate inlet ports, for example when a
bioreactor system is
used.
Other molecules can be added to the culture media, for instance, adhesion
molecules,
such as fibronectin or RetroNc:ctinT"" (commercially produced by Takara Shuzo
Co., Otsu
Shigi, Japan). The term fibronec;tin refers to a glycoprotein that is found
throughout the body
and its concentration is particularly high in connective tissues where it
forms a complex with
collagen. RetroNectinT"" may bE; used at a concentration range from about 0.1
p g/ml to about
1000 pg/ml. A more prefered range is about 0.2 pg/ml to S00 pg/ml and a most
prefered
range is about U.4 pg/ml to 100 ~cg/ml.
HDAC-I compounds arc: also included in the culture medium and these compounds
may be added individually or v1 combination, either prior to gene delivery or
during gene
delivery in an amount and under suitable conditions to allow stem cell self
renewal divisions.
The following compounds are r~spresentative examples of HDAC-I that may be
employed in
the present invention.
The microbial metabolite known as trichostatin A (TSA) is a HDAC-I (Yoshida,
et al.,
:?0 BioEssays, 17:42 - 429 ( 1995)). See EP 0 331 524 B 1 for methods of
preparation of TSA.
Other compounds related to TSA include butyrate, particularly sodium n-
butyrate; and hybrid
polar compounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA) and m-
carboxycinnamic acid bishydroxamide (CBHA). These compounds have two polar
groups
separated by an apolar S- to 6- carbon methylene chain. Additionally the
inhibitors appear to
:!5 cause the accumulation of acetylated histone H4 in cells in culture.
(Richon et al., Proc. Natl.
Acad Sci. 95:3003 - 3007 (1998)).
Another known microbial metabolite which functions as a HDAC-I is trapoxin, a
microbially derived cyclic tetrapeptide containing two L-phenylalanines,
specifically Trapoxin
30 A (TPX). (See Yoshida, et al., BioEssays, 17:42 - 429 (1995)). Not only is
the chemical
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structure of TPX completely different from TSA, the mode of inhibition is
totally different
from TSA and butyrate.
Cyclic tetrapeptide related compounds to TPX having the amino acid - 2-amino-8-
oxo-9, 10-epoxy-decanoic acid in their molecules are included as histone
deacetylase
inhibitors. The following compounds are mentioned: chlamydocin (Closse, et
al., Helv. Chim.
Acta 57: 533 - 545 ( 1974)), HC-toxin (Liesch, et al., Tetrahedron 38:45 - 48
( 1982)); Cyl-2;
and WF-3161 (Umehara, K. J. Antibiot 36: 478 - 483 ( 1983). The fungal
metabolite
depudecin, (See Kwon, et aL, Proc. Natl. Acad Sci. USA, 95:3356 - 3361 (
1998); and
t0 radicicoI are also mentioned as histone deacetylase inhibitors. W097/35990
discloses many
histone deacetylase inhibitor compounds and these are incorporated by
reference.
In a preferred embodiment, the HDAC-I is selected from the group of TSA, TPX,
chlamydocin, butyrate, particularly sodium n-butyrate, DMSO and HDAC-I analogs
thereof.
i.5 While the concentration range of the HDAC-I used will vary and will depend
on the specific
inhibitor, a preferred concentration range will be from about 0.001 nM to
about 10 mM, more
preferably from about 0.01 nM to about 1000 nM.
In a preferred embodiment, the histone deactylase inhibitor is (1) TSA
provided at a
2,0 concentration range from about 0.01 ng/mL to 1000 ng/mL, more preferably
from about 0.1
ng/mL to about 100 ng/mL, and most preferably from about 1.0 ng/mL to about 10
ng/mL,
(2) TPX, provided at a concentration range from about 0.001 nM to about 100
nM, more
preferably from about 0.01 nM to about 50 nM, and most preferably from about
0.1 nM to
about 1.0 nM; and (3) chlamydocin, provided at a concentration range from
about 0.001 nM
25 to about 100 nM, more preferably from about 0.01 nM to about 50 nM, and
most preferably
from about 0.1 nM to about 1.0 nM.
While specific histone deacetylase inhibitors have been defined and numerated
herein,
the scope of the invention includes a broad class of histone deacetylase
inhibitors including
30 compounds not enumerated herein. One means of determining whether a
compound not
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enumerated herein falls within the class of histone deacetylase inhibitors
includes but is not
limited to using standard enzymatic assays derived from measuring the ability
of an agent to
inhibit catalytic conversion of a substance by the subject protein. In this
manner, inhibitors of
the enzymatic activity of histone deacetylase proteins can be identified. (See
Yoshida, et al., J
Biol Chem. 265:17174-17179 (1990)). Compounds may be isolated by screening for
detransforming activity using oncogene-transformed cells ( Kwon, et al., Proc.
Natl. Acad
Sci. USA, 95:3356 - 3361 ( 1998). Furthermore, reference is made to Waterborg,
et al.,
Analytical Biochem. 122:313-318 {1982) for an assay that measures acetate
released by
deacetylation of lysine from a peptide derived from a N-terminal sequence of
histone H4.
This assay involves the extraction of tritiated acetyl groups with organic
solvent. A more
recent reference for HDAC assays is Kolle, et al., "Biochemical Methods for
Analysis of
Histone Deacetylases", Methods: A Companion to Methods of Enrymology 15:323-
331
(1998). Reference is also made t:o W097/35990 for disclosure of an assay used
to determine
if a compound is an HDAC-I. Inhibitors of HDAC may function by different
mechanisms and
mention is made of TSA and TP:~C. In one embodiment of the invention, an
effective amount
of a HDAC-I is used to promote self renewal division of stem cells and
particularly
hematopoietic stem cells in culture as compared to stem cells cultured under
essentially the
same conditions, but in the absence of HDAC-I in the culture. "Grown or
cultured under
essentially the same conditions" means the cells are exposed to the same
concentration of
2n growth supporting factors, the sane culture medium, and the same culture
period.
Once the cells are exposed to HDAC-Is, the culture period will vary and will
depend
on the culture conditions. Under i:he appropriate conditions cells potentially
could be cultured
indefinitely. However, in general, cells will be cultured from about 1 to
about 28 days,
2~> preferably from about 1 to about 14 days, more preferably from about 1 to
about 7 days.
However, cells may be exposed for 1 to about 5 days or for 1 to about 3 days.
Mention is
made that the culture period could be less than 1 day. While not meant to be a
limitation of
the invention, it takes in general cultured stem cells approximately 18 to 24
hours to undergo
self renewal divisions. However, after 5 days some of the cultured cells may
still be in the first
3G division. Therefore, cultured cells after 5 days may have completed various
numbers of self
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renewal divisions ranging from 1 to 7. After 5 days in culture the cells may
divide at the
approximate rate of one division per day or may return to a quiescent state.
In one
embodiment, the cells will be cultured for about 1 to about 5 days prior to
exposure to an
effective amount of a HDAC-I. However, the HDAC-I may be added upon the
initial
culturing. During this time frame, a gene delivery vehicle such as a vector
comprising a
nucleic acid sequence encoding a therapeutic gene or a marker gene may be
introduced into
the culture by means well known in the art.
Ex vivo stem cell self renewal may be measured by various different means.
Three
1o general ways include in vivo assays; in vitro assays; and analysis of
surface antigens. In vivo
assays include the use of the SCID-hu mouse system. {McCune, et al., Science
241: 1632 -
1639, 1988). In this system, human fetal bone fragments are implanted
subcutaneously into
severe combined immune deficient (SCID) mice. Once the implanted human fetal
bone
fragments are vascularized in the ;iCID-hu mouse, the marrow cavity can be
injected with a
test human cell population and analyzed for multilineage engraftment of the
donor cells.
Recently other models have been developed using the beige/nude/XID(bnx) mouse
and the
nonobese diabetic SCID (NOD-SCID) mouse. (Greiner, et al., Stem Cells 16:166-
177 (1998)
and Nolta et al., Proc. Natl. AcaGl. Sci. USA 93:2414-2419 (1996)). The
preferred way to
evaluate mammalian non-human stem cell activity is by transplantation into
irradiated hosts by
2o means well known to those skilled in the art.
In vitro systems for measurement of mammalian stem cell activity includes the
long
term culture initiating cell assay {LTCIC) and the cobblestone-area-forming
cell (CAFC)
assay. (Pettengell, et al., Blood 8~L:3653 ( 1994); Breems, et al., Leukemia
8:1095 ( 1994);
Reading, et al., Exp. Hem. 22:786 (Abst # 406) ( 1994); and Ploemacher, et
al., Blood
74:2755 ( 1989)). In the CAFC assay a sparsely plated cell population is
simply tested for its
ability to form distinct clonal outgrowths (or cobblestone areas) on a stromal
cell monolayer
over a period of time. This assay gives frequency readouts that correlate with
LTCIC and are
predictive of engraftment in in vivo assays and patients. A particularly
preferred CAFC assay
3o is described in Young, et al., Blood 88:1619 ( 1996).
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Flow cytometry can be used to subset hematopoietic cells from various tissue
sources
by the surface antigens they express. A combination of these assays may be
used to test for
HSCs. It is preferred that the SCII7-hu mouse model and the CAFC assay be used
to confirm
cells with stem cell function.
In one embodiment the invention concerns a model for measuring self renewal in
vitro
which includes labeling hematopoiietic cell populations particularly enriched
populations of
HSC with a division tracking dye. Division tracking dyes are fluorescently
tagged molecules
that bind stably to subcellular structures without apparent interference with
cellular function.
With each cell division, fluorescence intensity of the daughter cells is
halved relative to the
parent cell, allowing cell division to be tracked by flow cytometry. Many dyes
may be used
including but not limited to carbo~;yfluorescein diacetate succinimidyl ester
(CFSE); PKH26
(Young, et al., Blood 88:1619 ( 1996)); and PKH2 (Nordon, et al., British J.
Hematol. 98:528
- 539 {1997) and Traycoff, et al., ~;xp. Hematolo. 26:53-62 (1998)). Most
preferred is CFSE.
Antibodies conjugated to other fluorochromes and directed against surface
antigens
characteristic of HSCs are then used to allow simultaneous measurement of cell
division and
primitive phenotypes.
To test stem cell function after each division cycle in vitro, individual
populations can
be purified by flow cytametry and SCID-hu, and CAFC assays can be performed on
each
subset. The above list of assays used to measure functional compositions of
hematopoietic cell
populations is not meant to be limiting in any manner. One skilled in the art
may use other
known assays or combinations thereof.
In another preferred embodiment, the invention concerns a method of
genetically
modifying stem cells including contacting a population of stem cells with a
gene delivery
vehicle including a polynucleotide in the presence of an effective amount of a
HDAC-I. Gene
delivery or transfer can be mediated by methods well known in the art
including viral mediated
transfer of DNA or RNA; liposome mediated transfer; and other methods as
mentioned
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below. The term polynucleotide should be understood to include equivalent
terms such as
nucleotide sequence, nucleic acids and the like.
Retroviral vectors are partivcularly prefered gene delivery vehicles.
Retroviral vectors
enter the cell via its normal mechanism of infection or they can be modified
such that the
vector binds to a different cell surface receptor or ligand to enter the cell.
Additionally,
recombinant derived adenovirus (AD) vectors, particularly those that reduce
the potential for
recombination and generation o1E wild-type virus, have been constructed (see
W095/00655
and W095/11984). ADs are a relatively well characterized homogenous group of
viruses
including over 50 stereotypes (~~V095/27071; and Frey, et al., 1998 Blood
8:2781-2792).
ADs are easy to grow and do not require integration into the host cell genome.
Adeno-associated virus (AAV) has also been used as a gene delivery or transfer
system. (U.S. Pat. No. 5,693,531 and U.S. Pat. No. 5,691,176). They are small,
single-
stranded DNA viruses that can integrate into the genome of infected cells.
Recombinant AVV
vectors have been produced in hiigh titers which can transduce target cells at
high efficiency.
Herpes simplex virus (HSV) vectors are being considered by researchers for
gene therapy in
the transfer of gene to neural tissues. Various commercially available vectors
include: pSG,
pSV2CAT, and pXtl available from Stratagene and pMSG, pSVL, and pSVK3
available from
2o Pharmacia.
Vectors that contain both a promoter and a cloning site into which a
polynucleotide
can be operatively linked are well known in the art. Such vectors are capable
of transcribing
RNA in vitro or in vivo, and are commercially available from sources such as
Stratagene (La
Jolla, CA) and Promega Biotech. (Madison, WI). In order to optimize expression
and/or in
vitro transcription, it may be necessary to remove, add or alter 5' and/or 3'
untranslated
portions of the vectors to eliminate potentially inappropriate alternative
translation initiation
codons or other sequences that nnay interfere with or reduce expression,
either at the level of
transcription or translation. AltE:rnatively, consensus ribosome binding sites
can be inserted
3o immediately 5' of the start codon to enhance expression. Other non-limiting
elements which
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may be added to the vectors are enhancer elements, scaffold attachment regions
(SAR) and
matrix attachment regions (MAR), (See W097146687). Examples of these vectors
are
viruses, such as baculovirus, retroviruses, bacteriophages, cosmids, plasmids,
fungal vectors
and other recombination vehicles typically used in the art which have been
described for
expression in a variety of eukaryotic and prokaryotic hosts and may be used
for gene therapy
as well as for simple protein expression.
As mentioned above, preferred vectors are retroviral vectors, and particularly
preferred are amphotropic retroviral vectors. These vectors may include a
therapeutic gene or
lD a marker gene and a polynucleotide comprising the retroviral genome of part
thereof.
Reference is made to Coffin, et al., "Retroviruses", (1997) Chapter 9 pp: 437-
473 Cold
Springs Harbor Laboratory Press.. Retroviral vectors useful in the methods of
this invention
are produced recombinantly by procedures already taught in the art.
W094/29438,
W097/21824 and W097/21825 describe the construction of retroviral packaging
plasmids
and packaging cells lines. Retroviruses are subdivided into seven groups. Five
of these
groups represent retroviruses with oncogenic potential and the other two
groups are the
lentiviruses and spumaviruses. The most common retroviruses are those based on
the
Moloney murine leukemia virus I;MoMLV-vector). Other MoMLV derived vectors
include,
LMiLy, LINGFER, MINGFR anti MINT. Further vectors include those based on
Gibbon ape
2~D leukemia virus (GALV); Moloney murine sarcoma virus (MoMSV);
myeloproliferative
sarcoma virus (MPSV); murine embryonic stern cell virus (MESV), for example
MESV-
MiLy; murine stem cell virus (MSCV); and spleen focus forming virus (SFFV).
(Agarwal et
al., J. of Virology, 72:3720 ( 1998)). Non-limiting examples of 1enliviro1
derived vectors
include vectors based on human immunodeficiency virus (HIV-1 and HIV-2). New
vector
systems are continually being developed to take advantage of particular
properties of parent
retroviruses such as host range, usage of alternative cell surface receptors
and the like.
Particularly preferred vectors include DNA from a murine virus corresponding
to two long
terminal repeats and a package signal. In one embodiment the murine viral
vector is derived
from a MoMLV or a MSCV. Optionally, the vector will include one or more SAR
elements.
3~D However, the present invention is not limited to a particular retroviral
vector, but includes
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any retroviral vector wherein ~transduction of a population of HSCs is
enhanced in the
presence of HDAC-I over transduction of a population of HSCs under essentially
the same
conditions but in the absence of IHDAC-1 in the culture media.
In producing retroviral vectors constructs, the viral gag, pol and env
sequence will
generally be removed from the virus, creating room for insertion of foreign or
heterologous
DNA sequences. Genes encoded by foreign DNA are usually expressed under the
control of a
strong viral promoter in the long terminal repeat (LTR). Selection of
appropriate control
regulatory sequences is dependent on the host cell used and selection is
within the skill of one
in the art. Numerous promoters are known in addition to the promoter of the
LTR. Non-
limiting examples include the phage lamda PL promoter, the human
cytomegalovirus (CMV)
immediate early promoter; the jJ3 region promoter of the Moloney Murine
Sarcoma Virus
(MMSV), Rous Sarcoma Virus (RSV), or Spleen Focus Forming Virus (SFFV);
Granzyme A
promoter; CD34 promoter; and the CD8 promoter. Additionally, inducible or
multiple control
i.5 elements may be used. Plasmids containing retroviral genomes are widely
available from the
American Type Culture Collection (ATCC) and other sources known to those in
the art.
Such a construct can be packaged into viral particles efficiently if the gag,
pol and env
functions are provided in trans by a packaging cell line. Therefore, when the
vector construct
is introduced into the packaging cell, the gag-pol and env proteins produced
by the cell,
assemble with the vector RNA to produce infectious virions that are secreted
into the culture
medium. The virus thus produced can infect and integrate into the DNA of the
target cell, but
does not produce infectious viral particles since it is lacking essential
packaging sequences.
Most of the packaging cell lines currently in use have been transfected with
separate plasmids,
i;5 each containing one of the necessary coding sequences so that multiple
recombination events
are necessary before a replicatiion competent virus can be produced.
Alternatively, the
packaging cell line harbors a provirus. (The DNA form of the reverse-
transcribed RNA once
it integrates into the genomic DIVA of the infected cell). The provirus has
been crippled so
that although it may produce all the proteins required to assemble infectious
viruses, its own
RNA can not be packaged into virus. RNA produced from the recombinant virus is
packaged
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instead. Therefore, the virus stock released from the packaging cells contains
only
recombinant virus.
The range of host cells that may be infected by a retrovirus or retroviral
vector is
determined by the viral envelope protein. The recombinant virus can be used to
infect
virtually any other cell type recognized by the env protein provided by the
packaging cell,
resulting in the integration of the viral genome in the transduced cell and
the stable
incorporation of the foreign gene product. In general, murine ecotropic env of
MoMLV
allows infection of rodents cells, whereas amphotropic env allows infection of
rodent, avian
1o and some primate cells including human cells. Amphotropic packaging cell
lines for use with
MoMLV systems are known :in the art and are commercially available. Packaging
cell lines
include but are not limited to, PA12, PE501, PA317, PG 13, 'l'CRIP, RD 114,
GP7C-tTA-
G10, ProPak-A (PPA-6), PT67and FLYA13. (See Miller, et al., (1985) Mol. Cell
Biol.
5:431-437; Miller, et al., (1.986) Mal. Cell Biol. 6:2895-2902; Miller et al.,
(1989)
Biotechniques 7:980; Danos, et al. ( 1988) Proc. Natl. Acad. Sci. USA 85:b4b0-
6464; Rigg et
al., ( 1996) Virology 218:290 - 295; and Finer et al., ( 1994) Blood 83:43 -
50. Also reference
is made to WO 97121825 which discloses a method for obtaining a retrovirai
packaging cell
capable of producing retroviral vectors, particularly supernatants produced
from the
packaging cell lines ProPak are. taught. Recently, the G-glycoprotein from
vesicular stomatitis
2o virus (VSV-G) has been substiituted for the MoMLV env protein. (See Burns,
et al., (1993)
Proc. Natl. Acad Sci USA 90:8033-8037; and W092/14829). Xenotropic vector
systems
also exist which allow infection of human cells.
The vectors will contain at least one and preferably two heterologous genes or
gene
sequences; (i) a marker gene and (ii) a therapeutic gene to be transferred.
The following list
of potential genes that may be :incorporated into the vectors is given by way
of example and is
not meant to limit the inventior.~ in any manner.
A marker gene may be iinciuded in the vector for the purposes of monitoring
successful
3o transduction and for selection of cells into which the DNA has been
integrated. Non-limiting
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examples of marker genes include antibiotic resistance markers, such as
resistance to 6418 or
hygromycin. Additionally, negative selection may be used, for example, wherein
the marker is
the HSV-tk gene. This gene will make the cells sensitive to agents such as
acyclovir and
gancyclovir. Selection could also be made by use of a stable cell surface
marker to select for
transgene expressing stem cells by FACS sorting. The Neon (neomycin/G418
resistance)
gene is commonly used but any convenient marker gene whose sequences are not
already
present in the recipient cell can lne used. Further, non-limiting examples
include NGFR (nerve
growth factor receptor), GFP (the bacterial green fluorescent protein), DHFR
(a dihydrofolate
reductase gene which confers resistance to methotrexate), the bacterial hisD
gene, murine
1o CD24 (HSA), murine CDBa (l~~t), bacterial genes which confer resistance to
puromycin or
phleomycin, and ~i-galactosidase;.
The therapeutic gene may include genes or gene sequences effective in the
treatment
of adenosine deaminase deficiency (ADA); sickle cell anemia; recombinase
deficiency;
recombinase regulatory gene deficiency; HIV such as an antisense or trans-
dominant REV
gene or a gene carrying a herpes simplex virus thymidine kinase (HSV-tk)). The
therapeutic
gene may include gene sequences expressing the LDL (low-density lipoprotein)
receptor.
The therapeutic gene may also encode new antigens or drug resistant genes.
Further, the
therapeutic gene may encode a toxin or an apoptosis inducer effective to
specifically kill
2o cancerous cells, or a specific suicide gene to cancerous hematopoietic
cells may be included.
Therapeutic genes also encompass antisense oligonucleotides or ribozyme genes
useful for
translational suppression. The vector will usually comprise one therapeutic
gene. However,
more than one gene may be necc;ssary for the treatment of a particular
disease, and the vectors
used in the present invention may include more than one gene. Alternatively,
more than one
gene can be delivered using several compatible vectors. Depending on the
genetic defect, the
therapeutic gene can include rel;ulatory and untranslated sequences. For human
patients, the
therapeutic gene will generally 'be of human origin, although genes of closely
related species
that exhibit high homology and biologically identical or equivalent function
in humans may be
used if the gene does not produce an adverse immune reaction in the recipient.
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Nucleotide sequences far the therapeutic gene will generally be known in the
art or
can be obtained from various sequence databases such as GenBank. One skilled
in the art will
readily recognize that any therapeutic gene can be excised as a compatible
restriction fragment
and placed in a vector in such a. manner as to allow proper expression of the
therapeutic gene
in hematopoietic cells.
Methods of transduction include direct co-culture of cells with producer cells
(Bregni,
et al., (1992) Blood 80:1418-1422) or culturing with viral supernatant alone
with or without
appropriate growth factors and polycations (Xu, et al., Exp. Hemat. 22:223-
230). After viral
transduction, the presence of the viral vector in the transduced stem cells or
their progeny may
be verified by methods such as 1?CR. These techniques are well known in the
art.
The application of gene therapy using HSC is well known and the following
provides a
non-exhaustive list of diseases afar which gene transfer into HSCs is
potentially useful. These
diseases include bone marrow disorders, erythroid cell defects, metabolic
disorders and the
like. Particularly bone marrow transplantation should be enhanced by the
methods claimed
herein.
Genetically modified cells obtained by the methods described herein may be
further
used in autologous, xenogeneic: or allogenic settings. Autologous cells are
derived from an
individuals own tissue; xenogeneic cells are derived from a different species,
and allogenic
cells are derived from a genetically different individual of the same species.
The modified cells
may be administered in any physiologically acceptable vehicle, normally
intravascularly,
although they may also be introduced into bone or other convenient site which
cells may find
an appropriate site for regeneration and differentiation. Usually at least 1 x
105 cells may be
administered. Preferably, at l~;ast 1 x 106 or more cells may be introduced by
injection,
catheter or the like. If desired, factors such as TPO, IL-2, IL-3, IL-6, IL-
11, GM-CSF, G-
CSF, interferons, EPO and the life may also be included.
3o The methods provided by the present invention overcome deficiencies of
prior art
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methods of gene transfer by enhancing the transfer of genes into stem cells.
Gene integration
into stem cells by using vectors, particularly retroviral vectors requires
cell division. In
general, stem cell division results in differentiation. It is believed that
HDAC-Is may inhibit
differentiation during cell division, thereby increasing the CD34'Thy-1+ cell
content of the
transduced product.
The cells obtained as described above may be used immediately, expanded by
means
known in the art, or frozen at liquid nitrogen temperatures and stored for
long periods of
time, being thawed and capable of being used. The cells will usually be stored
in 10% DMSO,
50% FCS, and 40% RPMI 1640 medium. Once thawed, the cells may be further
expanded.
Methods of expansion of HSCs by use of growth factors and/or stromal cells
associated with
stem cell proliferation and differentiation are well known to those skilled in
the art. (US Pat.
No. 5,744,36 i )
The stem cell composition produced by the methods herein disclosed may be used
for
a variety of therapeutic means as disclosed in the literature. They can be
used to fully
reconstitute an immuno-compromised host such as an irradiated host and/or a
host subject to
chemotherapy. The cells may also be used for the treatment of genetic disease.
The cells may
be used in bone marrow transplants, where the cells may be freed of neoplastic
cells or other
:ZO cells that are pathogenic. The use of stem cells will minimize graft-
versus-host disease, and
may also be used to induce donor-specific immunlogic tolerance in the host.
Therefore an
embodiment of the invention vncludes a method of improving engraftment of
genetically
modified mammalian hematopoietic cells, particularly stem cells, including
exposing the
hematopoietic cells in a culture to an effective amount of a HDAC-I,
introducing a
:?5 polynulceotide encoding a therapeutic gene or a marker gene into the
cultured cells,
generating an increase in the number of genetically modified cells over that
in the absence of
exposure to the HDAC-I and ad,~ministering an effective amount of the
population of modified
cells to a subject. Preferably the; mammal and subject are human, and the
cells are enriched
hematopoietic stem cells, particularly CD34''Thy-1+ cells. In a preferred
embodiment, the
ao cells are transduced with a retroviral vector selected from MoMLV and MSCV
derived
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vectors and optionally include one or more SAR elements.
Additionally, a method a~f restoring hematopoietic capability in a subject is
claimed
comprising contacting a population of hematopoietic stem cells with an
effective amount of a
HDAC-I, transducing the HSCs .and administering an effective amount of a
population of the
transduced cells to the subject.
The practice of the present invention will employ, unless otherwise indicated
conventional techniques of cell biology, molecular biology, cell culture,
immunology and the
like which are in the skill of one in the art. These techniques are fully
disclosed in the current
literature and reference is made specifically to Sambrook, Fritsch and
Maniatis eds.,
"Molecular Cloning A Laboratory Manual, 2°° Ed., Cold Springs
Harbor Laboratory Press,
1989); the series Methods of Enzymology (Academic Press, Inc.); and
Antibodies: A
Laboratory Manual, Harlow et al.., eds., (1987).
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 cited herein are hereby incorporated by reference in their
entirety.
2D The invention generally described above will be more readily understood by
reference
to the following examples, which are hereby included merely for the purpose of
illustration of
certain embodiments of the present invention and are not intended to limit the
invention in any
way.
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EXPERIMENTAL
Ex~~le 1 - human cells:
Cells:
Following informed consent, leukaphersis samples are obtained from normal
donors
mobilized with ?.5 or 10 ~tg/kg/day of G-CSF for 5-6 days. CD34+ cells are
enriched from
leukaphersis samples at SyStemi:K using an Isolex 300SA or 300I (Baxter
Healthcare Corp.,
Deerfield, Illinois).
Purification of CD34+Thy-1+ cells from fresh MPB:
To select for CD34+'Thy-1+ cells by flow cytometry, anti-CD34, (PR20, SyStemix
Inc.,
Palo Alto CA) is directly conjugated to CY-5; and anti-human Thy-1 (PR13
SyStemix Inc.) is
directly conjugated to phycoerythrin (PE). Purified mouse IgGI (Sigma, St.
Louis, MO) is
directly conjugated to PE or Cy5 and used for isotype controls. Isolex-
selected MPB CD34+
cells are resuspended at 10' cel)s/mL in the staining buffer (SB) consisting
of Iscove's
modified Dulbecco's medium (IMDM) without phenol red (JRH Biosciences, Lenexa,
KS),
2% fetal bovine serum (FBS) (Gemini Bioproducts, Calabasas, CA), 0.1% heat
inactivated
human gamma-globulin (Gamimune) (Miles Inc., Elkhart, IN) and 10 mM HEPES (JRH
Biosciences). Cells are stained with anti-CD34-CY5 (5 pg/mL) and anti-Thy-1-PE
(25 It
a0 g/mL) or appropriate isotype controls, IgG 1-CY5 and IgG 1-PE, for 30
minutes at 4°C,
washed and resuspended in cold SB at 5x106 cells /mL. Propidium iodide (PI)
(Boehringer
Mannheim Biochemicals, Indianapolis, IN) is added at 1 ~tg/mL to detect
nonviable cells.
CD34''Thy-1+ and CD34~"Thy-1- cell populations are sorted on a Becton
Dickinson FACStar
Plus TM equipped with 5W argon laser (excitation 488nm) (Becton Dickinson, San
Jose,
:z5 CA). Sort regions for forward versus side scatter, PI, CD34, and Thy-1 are
established to
select live CD34'Thy-1+ or C'D34~Thy-1' cell populations. Sorted cell
populations are
reanalyzed to ensure clean separation of cell populations.
Cytokines and Cell Culture:
3o Recombinant human thrombopoietin (TPO) is obtained from R & D Systems,
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Minneapolis, MN. Recombinant Flt3 ligand (FL) and c-kit ligand (KL) are
produced at
SyStemix Inc., Interleukin (IL)-6 and leukemia inhibitory factor (LIF) are
obtained from
Novartis Inc., Basel, Switzerland. Cells are cultured for approximately 112
hours at 2 x 105
cells/mL (CD34+ cells) or 5 x 1.05 cells/mL (CD34+'Thy-1+ cells) in 6 or 24
well flat bottom
plates (Corning Costar Corp., Cambridge, MA) at 37 °C in a humidified
incubator with
approximately 100% saturation.. Cells are cultured in culture medium (CM) [X-
Vivo-15
medium, (BioWhittaker, Walke;rsville, MD) containing 1% bovine serum albumin
(BSA)
(Sigma)], supplemented with thc: cytokines: TPO (50 ng/mL), FL (100 ng/mL),
and KL (100
ng/mL). TSA is supplied at '.i nglmL; TPX is supplied at 0.25 nM and 0.5 nM
and
chlaunydocin is supplied at 0.25 nM or 0.50 nM. TSA is purchased from Wako
Bioproducts,
Richmond VA), and stored in absolute ETOH at -20 °C at a concentration
of lmg/mL. TPX
and chlaunydocin are provided lby Novartis Pharmaceuticals Corporation, East
Hanover, NJ
and stored in DMSO at -20 °C apt a concentration of lmM.
As shown in Figure l,. when CD34+ cells are cultured for 5 days with growth
supporting cytokines (TPO, FL and KL) and the HDAC-inhibitors TSA, TPX, or
chlamydocin, there is a substantial increase in the number of Thy-1+ cells
over cultures which
do not contain HDAC-inhibitors. Among the HDAC-inhibitors tested, chlamydocin
induced
the greatest increase, an average of 7 fold. These results are the average of
four experiments,
and the error bars show the standard deviation from the mean.
Isolation of cell populations poas culture:
Post culture cells are harvested from tissue culture wells, enumerated, and
stained with
anti-CD34-Cy5 and anti-Thy-l-PE or the appropriate isotype controls as
described above.
Cell populations are isolated with a FACStar Plus flow cytometer as described
above for
sorting pre-culture cells. Sorted cell populations are reanalyzed to ensure
that a majority of
events for each population falls. within each sort region, and to ensure that
contamination of
the Thy-1 populations by Thy-1~ cells is less than 5%.
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Cobblestone-area forming cell (CAFC) assay:
Cell populations are plated at limiting dilution (from 100-0.78 cells/well, 24
wells per
dilution) on pre-formed murine stromal monolayers, as described in Young et
al., Blood
88:1619 ( 1996). The culture medium consists of a mixture of equal portions of
RPMI and
IMDM medium (JRH BioSciences, Woodland, CA), 1mM sodium pyruvate (JRH
BioSciences), 5 x 10'5 M 2-mercaptoethanol (Sigma, St. Louis, MO), 10% FBS
(Hyclone,
Logan, UT), and cytokines, LIF (50 ng/mL) and IL-6 (10 ng/mL). Cultures are
fed at weekly
intervals by replacing half of ~~the medium, and scored at week 5 for
cobblestone area
;t0 formation. CAFC containing wells are scored microscopically, and their
frequencies estimated
statistically as described below.
A frequency readout is f;enerated from the numbers of positive wells in each
row of
the CAFC assay limiting dilution series using a SAS statistical analysis
program incorporating
U5 the maximum likelihood estimate method and the chi-square test of linearity
(Biostatistics
consulting, Palo Alto, CA). Limiting dilution readouts are considered to fit a
linear model if
the conservative chi-squared test of linearity was greater than 0.05. The
significance of
differences between CAFC frequency readouts is determined using Anova
(Microsoft), where
P less than 0.05 is considered to be significant.
:!o
As shown in Figure 2A, the number of Thy-1'' cells and the total CAFC activity
among
the Thy-1+ fraction of cells increased 2 to 3 fold in cultures containing
chlamydocin, versus
cultures without chlamydocin. 7.'otal cell numbers are the same in cultures
with or without
chlamydocin. However, total CAFC activity in the chlamydocin containing
cultures is
;!5 increased two-fold. (Figure 2B). The results indicate that stem cell
replication or self
renewal, measured by Thy-1+ ptuenotype as well as function is increased by
exposing cells to
HDAC-I during culture.
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SCID-hu bone assay:
The SCID-hu bone assay as described in Luens et al., Blood 91:1206 (1998) is
utilized. The hosts are C.B. 17 scid/scid mice implanted with human fetal
bones (Average
gestation age 22 weeks) at 8-10 weeks prior to use in assays. Starting donor
cell populations
for SCID-hu assays are CD:34 selected as described above. Post-culture donor
cell
populations are purified by flow cytometry for expression of the Thy-1 antigen
as described
above. The hosts are whole body irradiated (400 rads), then injected with
5,000 or 15,000
cultured Thy-1+ cells per human fetal bone graft. Eight weeks after the
injection, the mice are
sacrificed. Bone marrow is harvested from the grafts and stained with anti-
W6/32-PE for pan
human leukocyte antigen HLA class I major histocompatibility complex detection
versus an
appropriate FITC-conjugated antibody directed toward the HLA allo-type of the
donor cells.
The cells are analyzed on a FACScan (Becton Dickinson Immunocytometry System).
Grafts
showing a minimum of 1 % donor cells are scored as positive. The engraftment
rate is the
~s number of grafts positive for donor cells out of the number of grafts
injected. Table 1
illustrates that stem cell function is maintained on a per cell basis among
the Thy-1+ expressing
cells from cultures containing chlamydocin.
TABLE 1
Thy-1+ cells Dose Engraftment Rate
from cultures
(-) CHL 5,000 1/3
15,000 4/4
(+) CHL 5,000 2/3
15,000 3/3
CHL, is chlamydocin
Dose = cultured Thy-li~ cells per human fetal bone graft.
Engraftment Rate = the number of positive grafts for donor cells out of the
number of grafts
injected.
:25
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NOD SCID repopulation assay:
Trafficking to and enl;raftment in mouse bone marrow by human hematopoietic
cells
injected IV is measured by tlhe NOD SCID repopulation assay. Six to ten week
old NOD
SCID mice (Jackson derived" and bred at SyStemix) are irradiated with 350
rads. Human
MPB cells are injected into the tail vein or orbital sinus. Thy-1+ cells are
not repurifed after
culture for the present engraftment assays; instead, equal cell numbers from
the whole culture
are injected. Six weeks later l:he mice are sacrificed, and marrow cells from
the long bones of
the hind limbs are recovered. Cells are fluorescently labeled with anti-CD45-
APC which
detects human cells and analyzed on a FACS Calibur~. Results are illustrated
in Figure 6.
When cell dose is low and chl~amydocin is supplied in a 24 hour incubation,
the engraftment is
improved.
Division tracking dye labeling:
Isolex selected CD34+ cells are labeled with carboxyfluorescein-diacetate
succinimidylester {CFSE) dye (Molecular Probes Inc., Eugene, OR) at a cell
concentration of
3-5 x 106 /mL and a dye concentration of 1.25 p.M in IMDM without phenol red
in the dark,
at room temperature, for 10 minutes. The labeling is stopped by the addition
of 1l5 volume of
FBS and 10 fold volume of cold CM. Cells are washed and resuspended in CM. An
aliquot
of dye labeled cells is fixed with 1 % paraformaldehyde and stored at 4
°C to be used as a
marker for the fluorescence intensity of undivided cells post-culture.
Analysis is performed on
a FACSCalibur (Becton Dickinson, Immunocytometry System).
As shown in Figure 3, Thy-1 expression in culture after 5 days in the presence
of
chlamydocin is retained after at least 4 cell divisions, whereas it is more
rapidly lost with cell
division in cultures without chl,amydocin.
Analysis of cell phenotype post-culture:
Expression of CD34 and Thy-1 antigens is evaluated using anti-CD34-CYS and
anti-
Thy-1-PE as described above. FITC-conjugated monoclonal antibodies to human
CD2,
CD 14, CD 15, CD33, and CD 19 (Becton Dickinson, San Jose CA) are used to
evaluate
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expression of lineage antigens. Analysis is performed on a FACSCalibur.
Using the above described methods, it is determined that the expression of the
surface
lineage antigens CD2, CD 14, ~md CD 19 is negligible both before and after 5
day culture with
or without HDAC-inhibitors. 'The expression of CD 15 and CD33 was increased
post-culture,
but on a similar proportion of cells in cultures with or without HDAC-
inhibitors. Figure 4
shows that the proportion of Thy-1+ cells that express CD15 after 5 days of
culture is
approximately 30%, with or without HDAC-inhibitors. These results indicate
that although
expression of primitive antigens such as CD34 and Thy-1 is increased on cells
in cultures
containing HDAC-inhibitors, the expression of other surface antigens, such as
those
associated with differentiation, is not.
Example 2 - murine cells:
i5 Isolation of the murine stem cells:
Bone marrow is harvested from 4 week old BA.l mice (C57BIJKa.AKR/Jsys) and
enriched in Sca-1 expressing cells using a biotin-conjugated anti Sca-1
antibody (Pharmingen)
and positive selection using magnetic columns (Miltenyi Biotec Inc.). The Sca-
1 enriched cells
are further labeled with c-Kit-APC, Thyl.l-FITC, streptavidin-Texas Red and a
panel of PE
2o conjugated-lineage (Lip) antibodies directed against Mac-1, Gr-1, B220,
CD2, CD4, CDS,
CD8 (all antibodies purchased from Pharmingen). Propidium iodide (PI) is added
at 1 wg/ml to
exclude non-viable cells. The c;-Kit''Thy-1'~,in'°~-Sca-1'' subset,
which is highly enriched for
stem cell activity (Morrison a.nd Weissman, Immunity, 1:b61-673, (1994)) is
isolated by
FACS on a Vantage sorter (Bec;kton Dickinson, San Jose, CA).
Culture conditions:
The sorted cells are seeded in liquid culture at a density of 5x104 cellslml
in Xvivol5
media (BioWhittaker) in the presence of murine interleukin (mIL)-3 (10 ng/ml),
mIL-6 (10
nglml) and murine Stem Cell i:actor (mSCF) ( 100 nglml) (purchased from R&D
Systems).
3o TSA (Wako Bioproducts, Richmond VA) or chlamydocin (Novartis
Pharmaceuticals) are
added to the culture at concentrations ranging from 0.5 to 10 ng/ml and 0.25
to 2 nM,
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respectively. The cells are cultured 4 days in a 37°C incubator with 5%
COz in mufti-well flat
bottom tissue culture plates (either 9b well, 48 or 24 well plates depending
on the initial
number of cells seeded).
Analysis of cells post culture:
After 4 days of culture, the cells are harvested and an aliquot is stained
with Trypan
Blue to determine the number of viable and non-viable cells using a Neubauer
hemacytometer.
At least 200 total cells are scored. A portion of the cultured cells
(equivalent to at least 40
000 viable cells) ares then stained as described above. The profile of c-Kit ,
Thy-1, Lin and
1o Sca-1+ expression is then deternnined by FACS using a Vantage sorter. The
limits of the gates
used to determine the percentage of cells that fall into the c-Kit+'Thy-
1'"Lin'°'-Sca-1+ subset is
defined as the frontiers separating the bimodal population for each stain
observed separately.
These limits are set with cells treated with mIL-3, mIL-6 and mSCF alone. The
number of c-
Kiti'Thy-1+Lin'°'-Sca-l; cells preaent in the culture is calculated by
multiplying the proportion
of cells in the c-Kit'"Thy-1+L,in'°'~Sca-1+ subset by the total number
of viable cells present in the
culture.
Results from a representative experiment are summarized in Table 2 and
illustrated in
Figures SA, SB and SC.
TABLE 2
HDAC-I added Viable: cell % of non viable % of KTLS Fold expansion
(x1173) cells of KTLS cells
None 1 9:50 16% 8% 3.1
TSA 610 6% 58% 7.2
(3ng/ml)
Chlamydocin 450 3% 57% 5.0
(1nM)
KTLS is c-Kit'Thy-1'Lin'°''Sca-1+
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In this experiment SOxlO'' sorted c-Kit;'Thy-1'°Lin'd'Sca-1+(KTLS)
cells are cultured in
lml of X vivol5 media supplemf;nted with mIL-3, mIL-6 and mSCF in a 24 well-
plate. When
indicated TSA (3ng/ml) or chlamydocin (1nM) is added to the culture. After 4
days, the cells
are collected for cell count and FACS analysis. The number of viable cells
recovered after 4
days of culture is diminished 2 to 4 fold by the addition of HDAC-I to the
growth-media.
Compared to cells grown in the absence of HDAC-I, the vast majority of cells
treated with
TSA or chlamydocin remains Lizi negative (93 or 88% versus 55%) and expresses
high levels
of Thy-1 (95-96% versus 30%) and Sca-1 (94 or 88% versus 46%). A higher
percentage of
i.0 cells grown with either HDAC-I also remain positive for c-Kit compared to
cells cultured
without HDAC-I (67 and 73% versus 51%). While only 8% of the cultured cells
retained the
stem cell phenotype (c-Kit'Thy-1'"Lin'°'-Sca-1+) when grown in the
absence of HDAC-I, these
cells represent 58 and 57% of thn cells cultured with TSA or chlamydocin,
respectively. Over
the 4 day-culture period, the c~-Kit~T'hy-1'°Lin~'~-Sca-1+cells grown
without the addition of
HDAC-I underwent a 3.1-fold expansion while those cultured with TSA or
chlamydocin
underwent a 7.2- or 5.0-fold expansion, respectively.
Figures SA, SB and SC illustrate the profile of Lin, Thyl, cKit and Sca-1
which are
analyzed after 4 days of culture in growth media alone (A) or media
supplemented with 3
2,0 ng/ml of TSA (B) or 1nM of chlamydocin (C). The limits of the regions used
to calculate the
percentage of cells that fall into each gate are depicted on the dot plots.
The nonviable cells
are excluded from the analysis.
Example 3 - RetroNectin~ Transduction:
2.5
Retroviral Infection:
Following informed consent, as described above in example 1, the CD34'' of
CD34'Thy-1+ enriched cells are cultured at 106 cells per ml in 5 mL cultures
in X-Vivo-15
medium (BioWhittaker, Walkerswille, MD) for 48 hours at 37°C in 5% CO2.
The cultures are
30 supplemented with the cytolcines: TPO ( 100 ng/mL), (R&D Systems,
Minneapolis, MN); KL
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( 100 ng/mL), and FL ( 100 ng~mL), SyStemix, Palo Alto CA. The histone
deacetylase
inhibitor, chlamydocin is supplied to the cultures at 0.50 nM per mL for 72
hours.
Chlamydocin is provided by Novartis Pharmaceuticals Corporation, East Hanover,
NJ and is
stored in DMSO at -20 °C at a concentration of lmM.
Non-tissue culture-treated plates (Falcon, Lincoln Park, NJ) are coated at 2
~g per
cm2 of fibronectin fragment CH-296 (FN) (BioWhittaker, Walkersville, MD) and
incubated
for 2 hours at 37°C. Plates are blocked with 2% human serum albumin in
phosphate buffered
solution (HSAIPBS) for 30 minutes. This is followed by washing with 2.5% Hepes
buffer and
Hanks Balanced Salt Solution (HBSS).
After 48 hours, cultured cells are centrifuged for 10 minutes at 1200 rpm at 4
°C and
resuspended in the same medium as described above. The cells are added to FN-
coated plates
containing an equal volume of rexroviral supernatant for 20 hour culture at
37°C in 5% C02
without polybrene or protamine sulphate. (Lishan SU et al., 1997,
Hematopoietic Stem Cells-
Based Gene Therapy for Acquired Immunodeficiency Syndrome: Efficient
Transduction and
Expansion of RevMlO in Myeloid Cells In Vivo and In Vitro. Blood:Apr. 2283 -
2290).
The Moloney murine leukemia virus (MoMLV) vector is prepared from a ProPAk
(PP-A.6) packaging cell line (Forestell, et al., 1997, Novel Retroviral
Packaging Cell Lines:
Complementary Tropisms and Improved Vector Production For Efficient Gene
Transfer,
Gene Therapy, 4:600-610). The vector comprises - LTR-NGFR-SV40Neo-LTR- wherein
LTR is the viral long terminal repeat; NGFR is the truncated human nerve
growth factor
receptor, SV40 is the SV40 promoter, and Neo encodes 6418 resistance. (Miller,
et al., 1989,
Improved Retroviral Vectors for Gene Transfer and Expression. BioTechniques.
7:980 -990).
Cells are removed from the plates by gentle pipetting and centrifuged (as
described
previously). Cell pellets are resus~pended in X-vivo 15 medium plus 1% BSA.
Viable cells are
counted by trypan blue exclusion. After diluting, at a 1:10 ratio of cells to
trypan blue, cells
are placed in suspension in a hemacytometer. Dead cells are then determined by
their blue
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color. These methods are known t:o those skilled in the art.
FACS analysis of gene expression on CD34+, Thy-1 + and total cells:
After transduction, cells are analyzed by FACS as described below. A subset of
the
~~ cells as described above are ;placed in X-vivo 15 culture medium containing
GM-
CSF, l Ong/ml; EPO, 2 U; IL-3, 10 ng/ml; IL-6, 10 ng/ml; LIF, 100 ng/ml; and
KL 100
ng/ml. Cultures (1.0 ml) are performed in 24-well tissue culture plates
(Falcon). At 72 hours,
cultured cells are harvested and stained with anti-CD34-APC (Becton Dickinson,
San Jose,
CA), anti-Thy-1-PE (SyStemix, Palo Alto CA), and anti-NGFR-FITC (Boehringer
Mannheim,
Indianapolis, IN) or appropriata~ isotype controls (Becton Dickinson, San
Jose, CA).
Fluorescence is analyzed on a FAGS Calibur (Becton Dickinson) using standard
techniques.
The anti-NGFR-FITC is conjugated at SyStemix. Results summarized in Table 3
demonstrate
the application of chlamydocin increased not only the number of cells
expressing Thy-1
antigen but also the number of Th;y~ NGFR+ cells.
15~
TABLE 3
NGFR Transgene Expression in Primitive Thy-1' MPB Cells After
2G Short-term Culture (3 Days) With TPO, FL, and KL and with or without
Chlamydocin
%NGFR+ CELL#X
OF THY-1+ 10E5 OF
THY-1+NGFR'
AVE SD AVE SD
(-) 11.60 3.9 0.69 0.4
CHL
(+) 11.:?0 3.7 1.68 0.8
CHL
CHL 12.'.33 5.9 1.56 0.6
+
BSA
Fold 2.44
Increase
(+)
CHL
Footnote to Table 3.
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For (+) and (-) CHL, each value is the average (AVE) of 5 experiments.
For CHL + BSA each value is the average of 3 experiments.
SD = standard deviation.
CHL is Chlamydocin supplied at 0.5 nM/mL.
BSA is Borine Serum Albumin and is supplied at 1.0 %n.
Fold Increase = (+) average CHI. / (-) average CHL
In Vitro Assay for Transduction .of Primitive PHP:
1 o After approximately 72 hours of transduction, the cells are counted and
twenty
thousand cells per culture condition (+ or - chlamydocin) are plated on top of
SyS 1 marine
stromal cells (Young et al., Blood, vol. 87: 545 1996) in 2 wells in 24-well
plates (Corning
Science Products, Acton MA) for 5-week culture in the presence of exogenous
human IL-6
(20 ng/ml) and LIF ( 100 ng/mi). Plates are fed weekly by exchange of half the
medium
volume. The medium includes a mixture of 50/50 RPMI/IMDM plus 10% fetal calf
serum.
After 5 weeks in stromal culture, there is no significant difference between %
NGFR in CD34+
cells for cultures with or without chlamydocin. (Data not shown)
FACS Analysis of Gene Expression on CD34+ and total cells cultured on SYSI
Stroma:
Cells from above are harvested and filtered through 70mm cell strainers
(Falcon) to
remove stromal cell clumps. Viable hematopoietic cells are counted, and the
cells cultured for
3 days in X Vivo 15 medium containing GM-CSF, 10 nglml; EPO, 2U; IL-3, 10
ng/ml; IL-6,
10 ng/ml; LIF, 100 ng/ml; and K:L, 100 ng/ml. Cells are stained with anti-NGFR-
FITC and
anti-CD34-APC (Becton Dickinson). Cells are then analyzed on a FACS Calibur.
Further LTC-CFCAssays for SV40Neo on LTC-CFC can be performed'
Triplicates of 40,000 celas/ml from C are placed into methylcellulose colony
assays
(MethoCult, StemCell Technologies, Vancouver, Canada) with GM-CSF, 10 ng/ml;
EPO, 2U;
IL-3, 10 ng/ml; IL-6, 10 ng/ml; and KL, 100 ng/ml. Hematopoietic colonies are
scored after
12 - 14 days according to standard criteria known in the art.
Individual colonies (64) are placed into 50 ltl of lysis buffer as described
below.
Lysates are incubated overnight at 37 °C and heat inactivated at
95°C for 15 minutes, before
storage at -20°C. PCR assays ~~re performed to test transgene marking
with SV40Neo. B-
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globin is used as a positive control for the presence of DNA. PCR lysis buffer
solutions, A and
B, are mixed at a ratio of 1:1 with 144 mg Proteinase K. Lysis buffer solution
A includes 100
mM KCL, 10 mM Tris HCL, and 2.5 mM MgCl2. Lysis buffer solution B includes 10
mM
Tris HCL, 2.5 mM MgCl2,1 % 'rween, and 1 % NP40. The final concentration
obtained is 100
mg/ml Proteinase K. For PCR analysis, 10 p.l ( 1000 cells) and 5 p.l (500
cells) aliquots are
dispensed into thermocycle plates. Specific known primers and probes are used
for PCR:
PCR is carried out in 30 ~1 volumes, containing 10 mM Tris-HCL (pH 8.3), 1.5
mM
MgCl2, 50 mM KCL, 0.1666 m~Ivl each of dATP, dCTP, dGTP, dTTP, 0.833 ~tM of
SvNeo
to forward primer 1 and SvNeo rE:verse primer 2, 0.227 p.M B-globin forward
primer 1 and B-
globin reverse primer 2, and 1. unit of Taq DNA polymerase. The PCR program
can be
preformed on a Perkin-Elmer 9600 thermocycler: one cycle of 95 °C for 5
minutes and 40
cycles of 95 °C for 30 seconds, 62 °C for 30 seconds and 72
°C for 1 minute, followed by one
cycle of 72 °C for 10 minutes. PCR products are analyzed by gel
electrophoresis on 3
i5 agarose gels. These techniques .are well known in the art.
Example 4 - Spinoculation Trar~ a t'
Hematopoietic stem cells are purified by selection of CD34+ cells by an immune
2o affinity system from G-CSF mobilized peripheral blood obtained from healthy
volunteers using
standard practices and also as described in Example 1 above.
The purified CD34+ cells (3 x 106) are cultured in 7m1 Teflon cell culture
bags
(American Fluoroseal, Inc., Ga.itherburg, MD) for 48 hours in 1.5 ml serum-
free X-vivo 15
25 medium. The size of the Teflon bag used in an experiment is dependent on
the number of cells
to be cultured and subsequently transduced. The medium is supplemented with
hematopoietic
cell growth factors including '1,"PO, 200 ng/mL; FL, 200 ng/mL; IL-3, 40
ng/mL; IL-6, 40
ng/mL; and LIF, 200 ng/mL in the presence or absence of the histone
deacetylase inhibitor,
chlamydocin is supplied at 0.50 nM per mL.
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After 48 hours an equal volume ( 1.5 ml) of retroviral supernatant is added to
the
cultured cells. The retroviral vector and supernatant used in this example is
described above in
example 3. The cells are centrifuged at 3400 rpm (Sorval RD6000) oriented with
the flat side
of the bag placed at the bottom of the centrifuge bucket for 4 hours. The
cells are
resuspended by gentle pumping of the bag, and cells are returned to the
incubator. After 20
hours at 37 °C in 5% CO2, the cells are harvested from the bag, and
analyzed for stem cell
content (CD34+ and CD34''Thy-1'") as described above in Example 3.
to
TA_ BLE 4
Cell Recovery Post Transduction
Percent
Cell Recovery
Without With Chlamydocin
Chlamydocin
Mock: Transduced Mock Transduced
Range 83.9 - 115.760.0 - 158.683.2 - 144.381.4 - 151.1
Mean 104.5 95.9 118.5 113.
StDev 12.0 37.0 25.60 25.
n= 6 6 3 6
The data shown in Table 4 indicates that cell recovery after the transduction
process is
not effected by chlamydocin.
:ZO TABLE 5
The Effect of Chlamydocin on Stem Cell (CD34' Thy-1')
Content Post Transduction at Day 3
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% CD34'
Thy-1'
Starting Post Transduction
(Input)
(-) Chlamydocin(+) Chlamydocin
Range 40.8 - 24.9 - 44.6 49 - 79.8
G9.8
Mean 54.7 38.6 67.1
StDev 9.4 7.6 11.5
The mean represents the average of six experiments.
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The data provided in Tables 4 and 5 are used to calculate the absolute number
of HSC
{CD34+ Thy-1+) and the results are illustrated in Table 6. Yields are per 1 x
106 cells at the
start of transduction. The results suggest that there are approximately 2 fold
more HSCs in
the populations transduced with chlamydocin as compared to cells transduced
without the
chlamydocin. Cells transduc~ed in the presence or absence of chlamydocin
showed equivalent
content of CD34+ cells {99 -+-/-1.4%) after the 3 day transduction process.
TABLE
Calculation of Chiamydocin Effect on Stem Cell
1o Content Post Transduction
# of CD34'Thy-1+ % of input
cells
Input (starting)547000 100
(-) Chlamydocin370000 G7.6
(+) Chlamydocin758000 138.6
TABLE 7
Transgene Expression on HSC
% Cells
Expressing
NGFR
Total CD34+ CD34+Thy-1+
Cells
(-) {+) {-) {+) {-) (+)
CHLAM CHLAM CHLAM CHLAM CHLAM CHLAM
Range 8.7-19.37.4-28.4 8.8-19.8 7.6-29.4 5.0-15.15.0-27.2
Mean 13.1 18.1 13. S 18.7 9.1 15.9
StDev 3.9 7 4 7.3 4 7.9
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TA
Relative Effect of Chlamydocin on Thy Content
and Thy' Cells That Express Transgene on Day 3
As Compared to No Chlamydocin
Value Relative
to (-) Chlamydocin
% CD34'Thy-1+ % Thy-1+ expressing
NGFR
Range 1.63 - 1.96 0.93 - 2.43
Mean 1.75 1.71
StDev 0.12 0.45
Footnote to Table 8: 1.75 x 1.71 = approximately 3 fold increase
in the # of Thy+ c;ells that express the transgene in the infusion product.
The mean represents six experiments.
Example 5 - Dose Response F~~r Chlamydocin:
CD34+ selected cells are transduced by spinoculation with the MoMLV vector as
described in Example 3. The results shown in Table 9 illustrate chlamydocin
operates in a
dose dependent manner in increasing the stem cell content (CD34''Thy-1'") of
the ex vivo
transduced CD34+ cells derived from mobilized peripheral blood. The data
indicates that the
optimal concentration is between O.SnM and l.OnM for cells incubated for three
days.
2o Additionally, the results illustrate there is an increase in the stem cell
content relative to the
starting CD34+ population in the presence of 0.5 nM Chlamydocin.
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T. ABLE 9
Post Transduction Stem Cell Content
CHL Concentration CD34'Thy-1' Cells
(nM ) lo
0.0 16.2
O.:l_ 21.3
0.25 30.1
0.'i 54.0
1.0 48.8
Footnote to Table 9. The results are the averages of two experiments.
The CD43+'Thy-1~' cell content at the start of culture (Day 0) was 37 %.
