Note: Descriptions are shown in the official language in which they were submitted.
WO 90/15863 PCI/US90/03352
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ISOI,ATION GROWTEI AMD DIFFERhrNTIATION
5OF E~UMAN M~ISCLE CELLS
INTRODUCTION
Technical Field
The field of this invention is the development
of myoblasts for use in the treatment of neuromuscular
disease and for transformation for cellular therapy of
diseases of diverse etiology.
Backqround
Myoblasts are precursor cells of the mesoderm
that are destined for myogenesis. The determined
myoblasts are capable of re~ognizing and spontaneously
fusing with other myoblasts leading to the production
of a differentiated myotube. The multinucleated
myotube no longer divides or synthesizes DNA but
produces muscle proteins in large quantity. These
include constituents of the contractile apparatus and
25 specialized cell-surface components essential to
neuromuscular transmission. Eventually, the
differentiated muscle cell exhibits characteristic
striations and rhythmic contractions. A further step
in this pathway is maturation; the contractile ~`
apparatus and muscle at different stages of development
contain distinct isoforms of muscle proteins such as
myosin and actin, encoded by different members of
multigene families.
Methods have been developed for production of
myoblasts from fetal and adult tissue. The success of
these methods suggests that it is possible to generate
large volumes of myoblasts from adult muscle tissue
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that are substantially free o other cells. The
myoblasts have the potential for being used in a
variety of ways. First, the myoblasts may serve for
the treatment of various diseases associated with
genetic defects involving muscle tissue. The myoblasts
may also be found to be useful as vehicles for cell-
therapy, where one or more genes may be introduced into
the myoblasts to provide a product of interest.
Relevant Literature
Blau and Webster, Proc. Natl. Acad. USA (1981)
78:5623-5627 describe isolation and cloning of muscle
cells for proliferation or differentiation of
individual clones. Blau, et al., Proc. Natl. Acad. USA
(1983) 80:4856-4860 describe a defect in the
proliferative capacity of myoblasts (satellite cells),
mononucleated precursors of mature muscle fibers, in
clonal analyses of cells cultured from Duehenne
muscular dystrophy patients. Blau, et al., Ex~. Cell.
Res. ~1983) 144:495-503 describe the production and
analysis of pure myoblast clones from biopsies of
patients with Duchenne muscular dystrophy. Blau, et
al., Science (1985) 230:758-766 describe the fusion of
muscle cells with non-muscle cells with activation of
muscle gene expression in the non-muscle cell type.
Webster, et al., Exp. Cell Res. (1988) 174:252-265
describe the purification of human myoblasts using a
fluorescence-activated cell sorter. ~am, et al., In
Vitro Cell. ~ Dev. 8ioloqy ~1988) _ :833-844, describe
a serum-free medium for clonal growth of human muscle
satellite cells. Webster, et al., Cell (1988) 52:503-
513, describe that Duchenne muscular dystrophy
selectively affects a subset of skeletal muscle fibers
specialized for fast contraction (see also the
references cited therein).
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SUMMARY OF THE INVENTION
Myoblast cells are produced in serum free or
low serum media for use in cell-therapy. The myoblasts
are capable of migrating, fusing into pre-existing
fibers, and may serve as carriers for genes introduced
as a result of transformation. The migration of
myoblasts across basal lamina allows for a reduced
number of injections for treatment of a variety of
diseases.
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DESCRIPTION OF TBE SPECIFIC EM~ODIMENTS
Methods and cells are provided for use in ~ -
cellular therapies for the treatment of diseases.
Methods are described for preparing clonally pure or
substantially enriched myoblasts in large amounts in
the absence or substantial absence of serum in the
nutrient medium. The resultinq cells may be used for
treatment of a variety of diseases associated with
muscle tissue, or other tissue where a soluble factor
is involved.
The cells which are employed are myoblasts
which may be obtained from tissue samples, which may
include fetuses, neonates or tissue from older humans.
These cells may be fresh or may be immediately frozen
after being taken from the patient, or may be clonal
cultures which are grown up from about 5 to 30
population doublings, and then stored frozen for use.
The cells are grown in the subject media in a cell
culture incubator (37C, 5% C02 in air, saturated
humidity), as the optimum conditions. Other conditions
may be employed, if desired. The chosen medium
provides for proliferation, without significant
differentiation. Thus, the medium retains the myoblast
level of maturation and, when desired, the myoblasts
may be introduced into an environment, where they will
differentiate and mature.
The medium comprises a source of the essential
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amino acids, inorganic salts, trace elements and
vitamins, as well as other organic components The
following table indicates what has generally been found
to be optimum, although as is known in the field,
various changes may be made to individual components
without deleteriously affecting the growth of the
myoblasts.
.
lQ TABLE
PreferredBroad Concen-
Concentrationtration Range
M/l M~l
~ULK INORGANIC SALTS
CaC12-2H2 1.60 x 10 310-2 - 10-4
RCl - 4.00 x 10 310-3 - 10-4
M~SO4-7H2O 1.00 x 10 310 2 _ 10 i
NaCl 1.10 x 10 10.05 _ 0.5
Na2HPO4-7H2O 5.00 x 10 410-3 - 10-4
TRACE ELEMENTS
CUSO4-5H2O 1.00 x 10 810 7 - 10 9
FeSO4-7H2O 3.00 x 10 610-5 - 1o~6
H2SeO3 3.00 x 10 810-7 - 1o~8
MnSO4-5H2O 1;00 x 10 910 8 _ 1o~10
Na2SiO3-9H2O 1.00 x 10 510-4 - 10-5
(NH4)6Mo724-4H2 3.00 x 10 910 8 _ 10 9
N~4VO3 5.00 x 10 910 8 _ 10 9
NiC12-6H2 3.00 x 10 710-6 - 10-7
ZnSO4-7H2O 3.00 x 10 710-6 - 10-7
B~FFERS, INDICATORS
~ND ~ISCELLANEO~S
Phenol Red ~Na salt) 3.3 x 10-610-5 - 1o~6
NaHCO3 1.4 x 10 25x10 2 _ 5x10 3
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VITAMINS
d-Biotin 3.00 x 10 ~ 10-7 _ 1o~8
Folinic Acid
(Ca salt)-5H2O 1.00 x 10 6 5Xlo~6 - sxlo~7
DL-alpha-Lipoic Acid 1.00 x 10 ~ 5x10 8 _ 5x10
Niacinamide 5.00 x 10 5 10-4 - 10-5
D-Pantothenic Acid
(Hemi-Ca salt) 1.00 x 10 4 5x10 4 - 5x10 5
Pyridoxine 'dCl 1.00 x 10 5 5x10 5 - 5x10 6
Riboflavin 1.00 x 10 8 5x10 8 _ 5x10 9
Thiamin HCl 1.00 x 10 5 5x10 5 - Sx10 6
Vitamin B12 1.00 x 10 8 5x10 8 _ 5x10 9
OTEER ORGANIC COMPONENTS
Adenine 1.00 x 10 6 5x10 6 _ 5x10 7
Choline Chloride 1.00 x 10-4sxlo~4 - 5x10-5
D-Glucose 5.55 x 10 3 10-2 - 10-3
myo-Inositol 1.00 x 10 4 Sx10 4 - 5x10 5
Putrescine- 2HC1 1.00 x 10 9 5x10 9 - 5x10 10
Sodium Pyruvate 1.00 x 10-35x10-3 - sxlo-4
Thymidine 1.00 x 10 7 5x10 7 - 5x10 8
.
In addition to the basic medium which will be
referred to as MCDB120 additional factors are
supplied. The first factor is dexamethasone in an
amount of 0.3 to 0.5 ~g/ml, preferably about 0.39 to
0.40 ~g/ml. Serum album~n, particularly bovine serum
albumin, will be employed in from 0.25 to 0.75 mg/ml,
preferably about 0.50 mg/ml. Epidermal growth factor
is employed in from about 5 to 15 ng, preferably about
10 ng/ml. Fetuin is employed in from abou~ 0.25 to 0.75
mg/ml, preferably 0.5 mg/ml. Finally, insulin,
conveniently bovine insulin, may be employed in from
about 150 to 200 ~g/ml, preferably about 180 ~g/ml.
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To grow the myoblasts, an inoculum is
introduced into the medium described above and the
cells grown under the conditions described. After
adding the inoculum, mild agitation is employed to
ensure uniform distribution of the cells ~or further
growth toward confluency.
Cells may be harvested in any convenient
way. Tissue may be dissociated for a total of 40-60
min by two or three successive treatments with 0.05%
trypsin - EDTA at 37C in a Wheatin graduated
trypsinzation flask with constant stirring. The cells
collected in the supernatant after each trypsin
treatment are pooled and cooled to 4C on ice. Horse
serum is added to a final concentration of 10%
(vol/vol) to terminate further protease activity. The
dissociated cells are then centrifuged (2 min, 25C);
the cell pellet is resuspended in conditioned media and
either plated in culture or frozen in liquid nitrogen
at a density of about 0.1 cm3 of tissue per ml.
In culture, the myoblasts may be transformed
in any of a wide variety of ways, including fusion,
transfection, infection, electroporation, ballistics or
the like. The particular method for introducing the
foreign DNA is not crucial to this invention.
Depending on the purpose for the introduction of the
DNA, there may be an interest in having directed
homologous or legitimate integration by recombination
or illegitimate recombination. See 5mithies, et al.,
(1985) Nature 317:230-235; ~homas and Capecchi (1987)
C _ 51:503-512 and Mansour, et al. (1988) Nature_ _
336:348-352. For directed integration, the gene o~
interest will be flanked by at least 50 bp on each side
of DNA homologous to the site for integration, usually
at least 100 bp, and the total of homologous DNA may be
as high as 10 kbp, usually not greater than about 5
kbp, where preferably the flanking regions will be of
about the same size.
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Regions for integration may include DNA
sequences associated with a particular muscular~
defect. Thus, the host myoblasts may be removed from
the host, transformed by homologous recombination, and
cells cloned and screened for homologous recombination
at the site of the defect. Alternatively, where a
naturally occurring inducible gene is involved, which
is normally supressed in a myoblast or mature muscle
tissue, one may provide for homologous recombination,
where the transcriptional initiation regulatory
sequence, e.g., promoter with or without an enhancer,
is modified to provide for a different basis for
induction or for constitutive transcription. Thus, the
myoblasts may then be used for expression of an
endogenous gene (native to the host) or heterologous,
which is normally not expressed in muscle tissue. For
example, one may wish to provide for expression of
cytokines, growth factors, colony stimulating factors,
interferons, surface membrane receptors, insulin or the
like. ~y modiying the transcriptional initiation
regulatory region, the myoblasts may provide for
constitutive production of the expression product or
alternatively or in combination, one may introduce a
receptor for the soluble product, which provides for
inducible transcription of a cellular, e.g.,
cytoplasmic, nuclear, etc., protein. ~y activating the
receptor, the myoblasts may be induced to produce the
expression product under the induction of the relevant
ligand.
Various vehicles or vector constructs may be
employed for the transformation of the myoblast
cells. Of particular interest for transfection or
infection are replication-defective viral vectors, DNA
virus or retroviral vectors, which may be introduced
into the cells. The vectors will normally be free of
any prokaryotic DNA and may comprise a number of
different functional sequences. As already discussed,
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one of the functional sequences may be a DNA region
comprising transcriptional and translational initiation
and termination regulatory sequences, an open reading
frame encoding the protein of interest, and may further
comprise flanking regions for site directed
integration. In some situations, as already indicated,
the 5'-flanking region will provide for homologous
recombination to change the nature of the
transcriptional initiation region. ~or exa~ple, the
presence or absence of an enhancer may be modified, to
provide for inducible transcription or non-inducible
transcription, to increase or decrease the level of
transcription, or the like. Similarly, the promoter
region may be modified, so as to be more or less
susceptible to induction, to increase or decrease the
level of transcription, or the like.
The structural gene which is employed may
result in an intracellular product, i.e., retained in
the cell, in the cytoplasm or organelle, e.g., the
nucleus, in transport to a membrane, either an
intracellular membrane or the cell membrane, or for
secretion by providing for the natural signal sequence
present with the structural gene or a signal sequence
which is not naturally present with the structural
gene. In some situations, where the soluble protein of
interest is a fragment of a larger protein, it may be
necessary to provide a signal sequence with such
protein, so that upon secretion and processing at the
processing site, the desired protein ~ill have the
natural sequence.
~ marker may be present for selection of cells
which contain the vehicle construct. Normally, the
marker will allow for positive selection, in providing
protection from one or more cytotoxic agents. For
example, kanamycin resistance may be employed, where
the cells may be selected with G418, dihydrofolate
reductase may be employed for resistance to
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methotrexate, and the like. The marker may be an
inducible or non-inducible gene, so that selection may
occur under induction or without induction.
The vector may also include a replication
origin and such other genes which are necessary for
replication in the host. The replication system
comprising the origin and any proteins associated with
replication encoded by the particular virus may be
included as part of a construct. Care must be taken in
selecting the replication system, so that the genes
which are encoded for replication do not provide for
transformation of the myoblasts. Illustrative
replication systems include Epstein-Barr virus.
Alternatively, replication defective vehicles may be
employed, particularly replication-defective retroviral
vectors. These vectors are described by Price, et al.,
Proc. Natl. Acad. Sci.(1987) 84:156-160 and Sares, et
al. EMBO J. ~1986) 5:3133-3142. The final vehicle
construct may have one or more genes of interest.
Either a cDNA gene or a chromosomal gene may be
employed. Of particular interest is to provide for at
least one intron, which may be present in the 5'-non-
coding region or in the coding region. It is found
that the presence of an intron enhances stability of
the messenger RNA.
Alternatively, cells may be transformed in
vivo by injection of replication-defecti~e viral
vectors, which are infectious. The vectors may be
introduced into retroviral producer cells for ecotropic
packaging. The cells are then collected, filtered and
concentrated by centrifugation and the viral stock may
then be injected into a site in vivo. Since it is
found that the myoblasts will migrate, relatively few
injections into the muscle fibers are required, since
the myoblasts will expand into adjacent regions.
Administration of the vector is conveniently
by injection in a physiologically acceptable medium,
WO90/1SX63 PCT/US90/03352
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such as water, saline, phosphate buffered saline, or
the like. The viral concentration will generally be
from about 105 ffu. Other additives which may be
present include polybrene. Usually, the injections
will be about 105 cells per cm3 of muscle tissue. The
trauma to the tissue may be substantially minimized by
having only a few injections in the region of
interest. Particularly, where a patient may have need
for extensive treatment, the desirability of having a
low number of injections in a particular area is
manifest.
The following examples are offered by way of
illustration and not by way of limitation.
EXPERIMENTAL
Vectors
Two B-galactosidase vectors were employed,
referred to as BAG and pMMuLVSVnlsLacZ. The two
vectors each contain B-galactosidase encoding sequences
under the transcriptional control of the MMuLV
promoter/enhancer or the SV40 early promoter. The
vectors are further characterized by neo~ and LacZ
genes, and in some cases 7 amino acid codons for the
SV40 large T nuclear localization sequences.
Supernatant from CREBAG2 or PA12~212-C2
retroviral producer cells made by transfecting LAG into
the ~CRE or pMMuLVSVnlsLacZ into the Y2 ecotropic
packaging cell lines that are recombination resistant
(Price, et al., 1987; Sanes, et al., 1986),
respectively was collected, filtered and concentrated
by centrifugation. Viral stock (50-200 ~1) was mixed
with charcoal particles and 10 ~M polybrene and
injected from a 26 gauge needle into the latero-dorsal
surface of anaesthetized Wistar rat hindlimbs. Animals
were allowed to develop for about two weeks, then fixed
by cardiac perfusion with 4% paraformaldehyde, 0.5%
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glutaraldehyde, 100 mM PIPES pH 7.4.
After 30-60 min lower hindlimbs were dissected
free of skin and placed in the same fixative overnight
at 4C, then in 30% sucrose phosphate buffered saline
(PBS) for 24 h at 4~C, frozen in freezing isopentane
and cut into serial 30 ~m sections on a cryostat.
Sections were post-fixed in 2% paraformaldehyde, washed
and stained in 1 mg/ml X-gal, 35 mM potassium ferri-
and ferrocyanide, 1 mM MgC12 in PBS overnight at 30C,
mounted in glycerol:PBS (9:1) and examined under bright
field.optics with a Zeiss Axiophot microscope for the
presence of the blue X-gal reaction product. Clusters
of muscle fibers stained blue were scattered throughout
the lower hind limb. Charcoal particles were generally
located between soleus and lateral ~astrocnemius and
were used to identify the site of infection. No
differences in the distribution or size of clusters of
labeled muscle cells was observed with either the BAG
or pMMuLVSVnlsLacZ vectors. The BAG vector was
injected at P9 (P = postnatal) and analyzed at P23 and
the pMMuLVSVnlsLacZ was injected at P16 and analyzed at
P29. In addition, clusters of labeled cells close to
the charcoal particles were not detectably different
from those several millimeters away, suggesting that
the injection did not perturb development of nearby
tissue.
It was found that with rats injected at 9 days
and analyzed at 23 days, the number of clusters
spanning multiple fibers was 16 of 25, while with rats
of 19 days analyzed at 35 days, the number of clusters
spanning was 1 of 1.
Clusters of stained muscle fiberc in the
lateral gastrocnemius muscle demonstrated that the
myoblasts could be infected and expressed B-galacto-
sidase even after fusion into multinucleate fibers.Many of the clusters observed represented clones
derived from single cells, some of the progeny of which
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WO90/15863 ~5~9~ 12 PCTtUS90103352
migrated across basal lamina of a given muscle fiber
a~d fused into adjacent muscle fibers. Since each
myoblast is associated with a single fiber at the time
of infection, the data indicate that myoblasts are
capable of migrating through the basal lamina from one
fiber to another. Moreover, since the majority of
infection events yielded clones spanning multiple
muscle fibers, migration appears to be a relatively
frequent event.
To demonstrate that each cluster of
B-galactosidase-positive cells in an infected rat leg
originated clonally from a single retrovirally infected
myoblast, a mixture of two vectors were injected that
generate distinct B-galactosidase staining patterns:
lS nuclear and cytoplasmic. To maximize the number and
size of clones, a mixture of the two vectors was
injected into P0 rat hindlimbs, a time when extensive
proliferation and fiber formation is occurring. In two
legs heavily infected wi~h 87 separate s-galactosidase-
positive clusters of which 23 contained cytoplasmic
B-galactosidase, only in two instances did fibers
adjacent to a cytoplasmically-stained fiber contain
nuclear staining. Thus, the frequency with which
adjacent myoblasts are infected independently is below
15% in animals infected at a level of 40-50 clones per
lower hindlimb. Hence, in the less heavily infected
hindlimbs, it is unlikely that more than one or two of
the clones are in fact derived from two or more
separate infections. The vast majority are clones
derived from a single infection event.
Employing the dual virus technique, whether
each labelled fiber arose from a separate infection
event was investigated by determining whether there was
a random distribution of cytoplasmic and nuclear stains
among all fibers labelled. The contrary was observed,
in that cytoplasmic and nuclear staining patterns were
segregated into distinct areas, each including a
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cluster of several fibers.
It is unlikely that new fibers formed during
the period of the investigation, since the number of
fibers apparently remained constant. The mean diameter
of fibers within clones was similar to the overall mean
fiber diameter for the muscle suggesting no preferen-
tial inclusion of small, newly-formed fibers within
multifiber clones. The high frequency of multifiber
clones compared with the maximum possible rate of new
fiber formation suggest that the myoblasts destined to
form new fibers would have had to have been highly
preferred for infection by the vector compared to the
bulk population of dividing myoblasts. The data
strongly support the migration of myoblasts through the
basal lamina as the mechanism by which transformed
myoblasts are included in different fibers.
It is evident from the above results that the
subject invention allows for the use of muscle forming
cells in the treatment of diseases associated with
muscle tissue or for production of soluble or other
proteins in a host. The myoblasts are capable of
forming new muscle tissue or participating in the
formation of fibers, where the cells may provide for
useful properties, correct defects, and the like. In
addition, the cells may be modified with markers, to
allow for selective advantage of the transformed cells
over the naturally present cells.
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All publications and patent applications cited
in this specification are herein incorporated by
reference as if each individual publication or patent
application were specifically and individually
S indicated to be incorporated by reference.
Although the foregoing invention has been
described in some detail by way of illustration and
example for purposes of clarity of understanding, it
will be readily apparent to those of ordinary skill in
the art in light of the teachings of this invention
that certain changes and modifications may be made
thereto without departing from the spirit or scope of
the appended claims.
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