U.S. patent application number 15/436545 was filed with the patent office on 2017-08-10 for cytoplasmic transfer to de-differentiate recipient cells.
The applicant listed for this patent is Advanced Cell Technology, Inc.. Invention is credited to Karen B. Chapman.
Application Number | 20170226475 15/436545 |
Document ID | / |
Family ID | 22494867 |
Filed Date | 2017-08-10 |
United States Patent
Application |
20170226475 |
Kind Code |
A1 |
Chapman; Karen B. |
August 10, 2017 |
CYTOPLASMIC TRANSFER TO DE-DIFFERENTIATE RECIPIENT CELLS
Abstract
Methods for de-differentiating or altering the life-span of
desired "recipient" cells, e.g., human somatic cells, by the
introduction of cytoplasm from a more primitive, less
differentiated cell type, e.g., oocyte or blastomere are provided.
These methods can be used to produce embryonic stem cells and to
increase the efficiency of gene therapy by allowing for desired
cells to be subjected to multiple genetic modifications without
becoming senescent. Such cytoplasm may be fractionated and/or
subjected to subtractive hybridization and the active materials
(sufficient for de-differentiation) identified and produced by
recombinant methods.
Inventors: |
Chapman; Karen B.; (Mill
Valley, CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Advanced Cell Technology, Inc. |
Alameda |
CA |
US |
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Family ID: |
22494867 |
Appl. No.: |
15/436545 |
Filed: |
February 17, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14302384 |
Jun 11, 2014 |
9580683 |
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15436545 |
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13617988 |
Sep 14, 2012 |
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14302384 |
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13366518 |
Feb 6, 2012 |
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13617988 |
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10831599 |
Apr 23, 2004 |
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13366518 |
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09736268 |
Dec 15, 2000 |
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10831599 |
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PCT/US00/18063 |
Jun 30, 2000 |
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09736268 |
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60141250 |
Jun 30, 1999 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 5/0602 20130101;
C12N 5/0606 20130101; C12N 2503/00 20130101; A61K 48/00 20130101;
C12N 2501/727 20130101; C12N 2501/00 20130101; C12N 15/873
20130101; C12N 2510/00 20130101; C12N 2517/10 20130101; A61K 35/12
20130101; A61P 43/00 20180101; C12N 5/16 20130101; C12N 2510/04
20130101; C12N 5/0607 20130101; C12N 2506/00 20130101; C12N 9/1241
20130101; C12N 15/79 20130101; C12N 5/0696 20130101 |
International
Class: |
C12N 5/0735 20060101
C12N005/0735; C12N 15/873 20060101 C12N015/873; C12N 5/074 20060101
C12N005/074 |
Claims
1. A method for producing a totipotent cell comprising: a)
transferring all or part of the cytoplasm of a donor cell into an
isolated recipient cell; and b) transferring a telomerase or a DNA
construct that provides for the expression of telomerase into the
recipient cell or recipient cell nucleus; wherein the donor cell is
at or near senescence.
2. The method of claim 1, wherein the donor cell is less
differentiated than the recipient cell or is an undifferentiated
cell.
3. The method of claim 1, wherein said donor cell is an oocyte or
an embryonic cell.
4. The method of claim 1, wherein the telomerase DNA under the
control of a regulatable promoter.
5. The method of claim 1, wherein said recipient cell is a
mammalian cell.
6. The method of claim 5, wherein said mammalian cell is derived
from a mammal selected from the group consisting of non-human
primate, human, rat, guinea pig, mouse, rabbit, dog, cat, hamster,
goat, cattle, sheep, horse, bison and buffalo.
7. The method of claim 6, wherein said mammalian cell is selected
from the group consisting of cardiac, lung, skin, liver, stomach,
intestine, neural, muscle, bone, cartilage, immune, pancreatic,
spleen, esophageal, and corneal cells.
8. The method of claim 1, wherein said recipient cell or recipient
cell nucleus is genetically modified prior, concurrent or
subsequent to the introduction of said cytoplasm.
9. The method of claim 10, wherein (a) said genetically modified
cells comprise several genetic modifications; or (b) said
genetically modified recipient cell or recipient cell nucleus
comprises a recombinant DNA that encodes a desired polypeptide.
10. The method of claim 1, which results in the increased life-span
of a mammalian recipient cell or recipient cell nucleus.
11. The method of claim 1, wherein said donor cell is of a
different species than the recipient cell.
12. The method of claim 11, wherein said donor cell is a non-human
primate oocyte or embryonic cell and the recipient cell is a human
somatic cell.
13. An in vitro method for producing an embryonic stem cell, the
method comprising: a) providing a nucleus isolated from a cell; b)
transferring part of the cytoplasm of a cytoplasm donor cell into
said nucleus; c) introducing a telomerase or a DNA construct that
provides for the expression of telomerase into said nucleus or the
cell from which said nucleus is isolated; and d) introducing said
nucleus into a cytoplast.
14. The method of claim 13, wherein the embryonic stem cell has an
increased life-span relative to the cell from which said nucleus is
isolated, and wherein the donor cell is at or near senescence.
15. The method of claim 13, wherein the cytoplasm is derived from
an oocyte or embryonic cell.
16. The method of claim 13, wherein the telomerase DNA under the
control of a regulatable promoter.
17. The method of claim 13, wherein the cell from which said
nucleus is isolated is a mammalian cell.
18. The method of claim 13, wherein said recipient cell or
recipient cell nucleus is genetically modified prior, concurrent or
subsequent to the introduction of said cytoplasm.
19. The method of claim 13, wherein said donor cell is of a
different species than the recipient cell.
20. The method of claim 19, wherein said donor cell is a non-human
primate oocyte or embryonic cell and the recipient cell is a human
somatic cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 14/302,384 filed Jun. 11, 2014, now issued as
U.S. Pat. No. 9,580,683; which is a continuation application of
U.S. application Ser. No. 13/617,988 filed Sep. 14, 2012, now
abandoned; which is a continuation application of U.S. application
Ser. No. 13/366,518 filed Feb. 6, 2012, now abandoned; which is a
continuation application of U.S. application Ser. No. 10/831,599
filed Apr. 23, 2004, now abandoned; which is a continuation
application of U.S. application Ser. No. 09/736,268 filed Dec. 15,
2000, now abandoned; which is a 35 USC .sctn.371 National Stage
application of International Application No. PCT/US00/18063 filed
Jun. 30, 2000, now expired; which claims the benefit under 35 USC
.sctn.119(e) to U.S. application Ser. No. 60/141,250 filed Jun. 30,
1999, now expired. The disclosure of each of the prior applications
is considered part of and is incorporated by reference in the
disclosure of this application.
BACKGROUND OF THE INVENTION
[0002] Field of the Invention
[0003] The present invention relates to methods for
"de-differentiating" and/or altering the life-span of desired
recipient cells, preferably human somatic cells. These methods have
application especially in the context of cell therapies and the
production of genetically modified cells.
[0004] Background Information
[0005] Nuclear transfer first gained acceptance in the 1960's with
amphibian nuclear transplantation. (Diberardino, M. A. 1980,
"Genetic stability and modulation of metazoan nuclei transplanted
into eggs and ooctyes", Differentiation, 17-17-30; Diberardino, M.
A., N. J. Hoffner and L. D. Etkin, 1984; "Activation of dormant
genes in specialized cells", Science, 224:946-952; Prather, R. S.
and Robl, J. M., 1991, "Cloning by nuclear transfer and splitting
in laboratory and domestic animal embryos", In: Animal Applications
of Research in Mammalian Development, R. A. Pederson, A. McLaren
and N. First (ed.), Cold Spring Harbor Laboratory Press.) Nuclear
transfer was initially conducted in amphibians in part because of
the relatively large size of the amphibian oocyte relative to that
of mammals. The results of these experiments indicated to those
skilled in the art that the degree of differentiation of the donor
nucleus was greatly instrumental, if not determinative, as to
whether a recipient oocyte containing such cell or nucleus could
effectively reprogram said nucleus and produce a viable embryo.
(Diberardino, M. A., N J. Hoffner and L. D. Etkin, 1984,
"Activation of dormant genes in specialized cells.", Science,
224:946-952; Prather, R. S. and Robl, J. M., 1991, "Cloning by
nuclear transfer and splitting in laboratory and domestic animal
embryos", In: Animal Applications of Research in Mammalian
Development, R. A. Pederson, A. McLaren and N. First (ed.), Cold
Spring Harbor Laboratory Press.)
[0006] Much later, in the mid 1980s, after microsurgical techniques
had been perfected, researchers investigated whether nuclear
transfer could be extrapolated to mammals. The first procedures for
cloning cattle were reported by Robl et al (Robl, J. M., R.
Prather, F. Barnes, W. Eyestone, D. Northey, B. Gilligan and N. L.
First, 1987, "Nuclear transplantation in bovine embryos", J. Anim.
Sci. 64:642-647). In fact, Dr. Robl's lab was the first to clone a
rabbit by nuclear transfer using donor nuclei from earlier
embryonic cells (Stice, S. L. and Robl, J. M., 1988, "Nuclear
reprogramming in nuclear transplant rabbit embryos", Biol. Reprod.,
39:657-664). Also, using similar techniques, bovines (Prather, R.
S., F L. Barnes, M L. Sims, Robl, J. M., W. H. Eyestone and N. L.
First, 1987, "Nuclear transplantation in the bovine embryo:
assessment of donor nuclei and recipient oocyte", Biol. Reprod.,
37:859-866) and sheep (Willadsen, S. M., 1986, "Nuclear
transplantation in sheep embryos", Nature (Lond) 320:63-65), and
putatively porcines (Prather, R. S., M. M. Sims and N. L. First,
1989, "Nuclear transplantation in pig embryos", Biol. Reprod.,
41:414), were cloned by the transplantation of the cell or nucleus
of very early embryos into enucleated oocytes.
[0007] In the early 1990s, the possibility of producing nuclear
transfer embryos with donor nuclei obtained from progressively more
differentiated cells was investigated. The initial results of these
experiments suggested that when an embryo progresses to the
blastocyst stage (the embryonic stage where the first two distinct
cell lineages appear) that the efficiency of nuclear transfer
decreases dramatically (Collas, P. and J. M. Robl, 1991,
"Relationship between nuclear remodeling and development in nuclear
transplant rabbit embryos", Biol. Reprod., 45:455-465). For
example, it was found that trophectodermal cells (the cells that
form the placenta) did not support development of the nuclear
fusion to the blastocyst stage. (Collas, P. and J. M. Robl, 1991,
"Relationship between nuclear remodeling and development in nuclear
transplant rabbit embryos", Biol. Reprod., 45:455-465.) By
contrast, inner cell mass cells (cells which form both somatic and
germ line cells) were found to support a low rate of development to
the blastocyst stage with some offspring obtained. (Collas P,
Barnes F L, "Nuclear transplantation by microinjection of inner
cell mass and granulosa cell nuclei", Mol Reprod Devel., 1994,
38:264-267.) Moreover, further work suggested that inner cell mass
cells which were cultured for a short period of time could support
the development to term. (Sims M, First N L, "Production of calves
by transfer of nuclei from cultured inner cell mass cells", Proc
Natl Acad Sci, 1994,91:6143-6147.)
[0008] Based on these results, it was the overwhelming opinion of
those skilled in the art at that time that observations made with
amphibian nuclear transfer experiments would likely be observed in
mammals. That is to say, it was widely regarded by researchers
working in the area of cloning in the early 1990's that once a cell
becomes committed to a particular somatic cell lineage that its
nucleus irreversibly loses its ability to become "reprogrammed",
i.e., to support full term development when used as a nuclear donor
for nuclear transfer. While the exact molecular explanation for the
apparent inability of somatic cells to be effectively reprogrammed
was unknown, it was hypothesized to be the result of changes in DNA
methylation, histone acetylation and factors controlling
transitions in chromatin structure that occur during cell
differentiation. Moreover, it was believed that these cellular
changes could not be reversed.
[0009] Therefore, it was quite astounding that in 1998, the Roslin
Institute reported that cells committed to somatic cell lineage
could support embryo development when used as nuclear transfer
donors. Equally astounding, and more commercially significant, the
production of transgenic cattle which were produced by nuclear
transfer using transgenic fibroblast donor cells was reported
shortly thereafter by scientists working at the University of
Massachusetts and Advanced Cell Technology.
[0010] Also, recently two calves were reportedly produced at the
Ishikawa Prefecture Livestock Research Centre in Japan from oviduct
cells collected from a cow at slaughter. (Hadfield, P. and A.
Coghlan, "Premature birth repeats the Dolly mixture", New
Scientist, Jul. 11, 1998.) Further, Jean-Paul Renard from INTRA in
France reported the production of a calf using muscle cells from a
fetus. (MacKenzie, D. and P. Cohen, 1998, "A French calf answers
some of the questions about cloning", New Scientist, March 21.)
Also, David Wells from New Zealand reported the production of a
calf using fibroblast donor cells obtained from an adult cow.
(Wells, D. N., 1998, "Cloning symposium: Reprogramming Cell
Fate--Transgenesis and Cloning," Monash Medical Center, Melbourne,
Australia, Apr. 15-16.)
[0011] Differentiated cells have also reportedly been successfully
used as nuclear transfer donors to produce cloned mice. (Wakayama
T, Perry A C F, Zucconi M, Johnsoal K R, Yanagimachi R., "Full-term
development of mice from enucleated oocytes injected with cumulus
cell nuclei", Nature, 1998, 394:369-374.)
[0012] Still further, an experiment by researchers at the
University of Massachusetts and Advanced Cell Technology was
recently reported in a lead story in the New York Times, January
1999, wherein a nuclear transfer fusion embryo was produced by the
insertion of an adult differentiated cell (cell obtained from the
cheek of an adult human donor) into an enucleated bovine oocyte.
Thus, it would appear, based on these results, that at least under
some conditions differentiated cells can be reprogrammed or
de-differentiated.
[0013] Related thereto, it was also recently reported in the
popular press that cytoplasm transferred from oocyte of a young
female donor "rejuvenated" an oocyte of an older woman, such that
it was competent for reproduction.
[0014] However, it would be beneficial if methods could be
developed for converting differentiated cells to embryonic cell
types, without the need for cloning, and the production of embryos,
especially given their potential for use in nuclear transfer and
for producing different differentiated cell types for therapeutic
use. Also, it would be beneficial if the cellular materials
responsible for de-differentiation and reprogramming of
differentiated cells could be identified and produced by
recombinant methods, thereby improving the efficiency of cellular
reprogramming.
OBJECTS OF THE INVENTION
[0015] Therefore, it is an object of the invention to provide novel
methods for "de-differentiating" and/or altering the life-span of
desired cells.
[0016] It is a more specific object of the invention to provide a
novel method for "de-differentiating" and/or altering the life-span
of a desired differentiated cell by introducing the cell or cell
nucleus with cytoplasm and then transplanting the de-differentiated
nucleus into a surrogate cytoplast such as from an ES cell of a
less differentiated cell, preferably an oocyte or blastomere, or
another embryonic cell type.
[0017] It is another object of the invention to alter the life-span
and/or to de-differentiate desired cells, typically mammalian
differentiated cells, prior, concurrent, or subsequent to genetic
modification.
[0018] It is another object of the invention to provide an improved
method of cell therapy wherein the improvement comprises
administering cells which have been de-differentiated or have an
altered life-span by the introduction of cytoplasm obtained from a
cell of a less or undifferentiated state, preferably an oocyte or
blastomere or placing nuclei from said somatic cell into a solution
containing an extract of the oocyte or blastomere embryo, or ES
cell or purified proteins from the same.
[0019] It is still another object of the invention to identify the
component or components in oocyte cytoplasm responsible for
de-differentiation and/or alteration of cell life-span, e.g., by
fractionation or subtractive hybridization, i.e., fractionation of
protein, RNA or DNA.
[0020] It is still another object of the invention to provide a
novel method of therapy, especially of the skin, by administering a
therapeutically effective amount of cytoplasm obtained from a
substantially undifferentiated or undifferentiated cell, preferably
an oocyte or blastomere, or the purified active components of the
same.
[0021] It is another object of the invention to provide novel
compositions for therapeutic, dermatologic and/or cosmetic usage
that contain cytoplasm derived from substantially undifferentiated
or undifferentiated cells, preferably an oocyte or blastomere, or
purified active components of same.
[0022] It is another object of the invention to provide cells for
use in cell therapy which have been "de-differentiated" or have an
altered life-span by the introduction of cytoplasm from a
substantially undifferentiated or undifferentiated cell, preferably
an oocyte or blastomere, or purified active components of same.
[0023] It is still another object of the invention to provide an
improved method of cloning via nuclear transfer wherein the
improvement comprises using as the donor cell or nucleus a cell
which has been de-differentiated and/or has had its life-span
altered by the introduction of cytoplasm from a substantially
undifferentiated or undifferentiated cell, or purified active
components of same, or cross-species NT where the purified active
component is expressed to facilitate reprogramming.
[0024] It is another object of the invention to rejuvenate nuclei
isolated from desired differentiated cells by contacting same with
cytoplasm from oocytes, blastomeres, ES, or other embryonic cell
types.
[0025] It is another object of the invention to provide screening
assays to identify proteins, or nucleic acid sequences that are
released from differentiated cell nuclei upon contacting with
cytoplasm, or fractions derived from oocyte cytoplasm from oocytes,
blastomeres, ES cells or other embryonic cell types, that are
involved in all reprogramming.
[0026] It is another specific object of the invention to provide
screening assays, e.g., differential or subtractive hybridization
to identify mRNAs that expressed in oocyte cytoplasm or in
embryonic cell types that are involved in cell programming.
SUMMARY OF THE INVENTION
[0027] The present invention provides novel methods for producing
cells, preferably mammalian cells and, most preferably, human cells
that have been de-differentiated and/or which have an altered
(increased) life-span by the juxtaposition of the donor cell with
cytoplasm from an undifferentiated or substantially
undifferentiated cell, preferably an oocyte or blastomere, or
another embryonic cell type. In a particularly preferred
embodiment, the present invention will be used to produce cells in
a more primitive state, especially embryonic stem cells or inner
cell mass cells.
[0028] The resultant cells are useful in gene and cell therapies,
and as donor cells or nuclei for use in nuclear transfer.
DEFINITIONS
[0029] "Ooctye"--In the present invention, this refers to any
oocyte, preferably a mammalian oocyte, that develops from an
oogonium and, following meiosis, becomes a mature ovum.
[0030] "Metaphase II Ooctye"--The preferred stage of maturation of
oocytes used for nuclear transfer (First and Prather,
Differentiation, 48:1-8). At this stage, the oocyte is sufficiently
"prepared" to treat an introduced donor cell or nucleus as it does
a fertilizing sperm.
[0031] "Donor Cell"--In the present invention, this refers to a
cell wherein some or all of its cytoplasm is transferred to another
cell ("recipient cell"). The donor cell is typically a primitive or
embryonic cell type, preferably an oocyte, blastomere, or inner
cell mass cell.
[0032] "Recipient Cell"--This refers to a cell into which all or
part of the cytoplasm of a donor cell, wherein such donor cell is
of a more primitive cell type relative to the recipient cell, is
transferred. This transfer can be accomplished by different
methods, e.g., microinjection or by contacting donor cells with
liposomal encapsulated cytoplasm or enucleating the donor cell and
incubating with cytoplasmic extract. Typically, the donor cell is
an oocyte, blastomere or inner cell mass cell, and the recipient
cell is a somatic cell, preferably a human somatic cell.
[0033] "Blastomere"--Embryonic, substantially undifferentiated
cells contained in blastocyst stage embryos.
[0034] "Embryonic Cell" or "Embryonic Cell Type"--In the present
invention, this will refer to any cell, e.g., oocyte, blastomere,
embryonic stem cell, inner cell mass cell, or primordial germ cell,
wherein the introduction of cytoplasm therefrom into a
differentiated cell, e.g., human somatic cell in tissue culture,
results in de-differentiation and/or lengthening of the life-span
of such differentiated cell.
[0035] "Cell Having Altered Life-Span"--In the present invention
this refers to the change in cell life-span (lengthening) that
results when cytoplasm of a more primitive or less differentiated
cell type, e.g., an embryonic cell or embryonic cell type, e.g.,
oocyte or blastomere, is introduced into a desired differentiated
cell, e.g., a cultured human somatic cell.
[0036] "Embryonic Stem Cell (ES Cell)"--In the present invention
this refers to an undifferentiated cell that has the potential to
develop into an entire organism, i.e., a cell that is able to
propagate indefinitely, maintaining its undifferentiated state and,
when induced to differentiate, be capable of giving rise to any
cell type of the body.
[0037] "Nuclear Transfer"--Introduction of cell or nuclear DNA of
donor cell into enucleated oocyte which cell or nucleus and oocyte
are then fused to produce a nuclear transfer fusion or nucleus
fusion embryo. This NT fusion may be used to produce a cloned
embryo or offspring or to produce ES cells.
[0038] "Telomerase"--A ribonucleoprotein (RNP) particle and
polymerase that uses a portion of its internal RNA moiety as a
template for telomere repeat DNA synthesis (U.S. Pat. No.
5,583,016; Yu et al, Nature, 344:126 (1990); Singer and
Gottschling, Science, 266:404 (1994); Autexier and Greider, Genes
Develop., 8:563 (1994); Gilley et al, Genes Develop., 9:2214
(1995); McEachern and Blackburn, Nature, 367:403 (1995); Blackburn,
Ann. Rev. Biochem., 61:113 (1992); Greider, Ann. Rev. Biochem.,
65:337 (1996).) The activity of this enzyme depends upon both its
RNA and protein components to circumvent the problems presented by
end replication by using RNA (i.e., as opposed to DNA) to template
the synthesis of telomeric DNA. Telomerases extend the G strand of
telomeric DNA. A combination of factors, including telomerase
processivity, frequency of action at individual telomeres, and the
rate of degradation of telomeric DNA, contribute to the size of the
telomeres (i.e., whether they are lengthened, shortened, or
maintained at a certain size). In vitro telomerases may be
extremely processive, with the Tetrahymena telomerase adding an
average of approximately 500 bases to the G strand primer before
dissociation of the enzyme (Greider, Mol. Cell. Biol., 114572
(1991).)
[0039] "Genetically Modified or Altered"--In the present invention
this refers to cells that contain one or more modifications in
their genomic DNA, e.g., additions, substitutions and/or
deletions.
[0040] "De-Differentiation"--In the present invention, this refers
to the changes in a differentiated cell, e.g., human somatic cell
in tissue culture, that result upon introduction of cytoplasm from
a more primitive, less differentiated cell type, e.g., an oocyte or
other embryonic cell.
[0041] "Totipotent"--In the present invention this refers to a cell
that gives rise to all of the cells in a developing body, such as
an embryo, fetus, an animal. The term "totipotent" can also refer
to a cell that gives rise to all of the cells in an animal. A
totipotent cell can give rise to all of the cells of a developing
cell mass when it is utilized in a procedure for creating an embryo
from one or more nuclear transfer steps. An animal may be an animal
that functions ex utero. An animal can exist, for example, as a
live born animal. Totipotent cells may also be used to generate
incomplete animals such as those useful for organ harvesting, e.g.,
having genetic modifications to eliminate growth of a head such as
by manipulation of a homeotic gene.
[0042] "Ungulate"--In the present invention this refers to a
four-legged animal having hooves. In other preferred embodiments,
the ungulate is selected from the group consisting of domestic or
wild representatives of bovids, ovids, cervids, suids, equids, and
camelids. Examples of such representatives are cows or bulls,
bison, buffalo, sheep, big-horn sheep, horses, ponies, donkeys,
mule, deer, elk, caribou, goat, water buffalo, camels, llama,
alpaca, and pigs. Especially preferred in the bovine species are
Bos taurus, Bos indicus, and Bos buffaloes cows or bulls.
[0043] "Immortalized" or "Permanent"Cell--These terms as used in
the present invention in reference to cells can refer to cells that
have exceeded the Hayflick limit. The Hayflick limit can be defined
as the number of cell divisions that occur before a cell line
becomes senescent, Hayflick set this limit to approximately 60
divisions for most non-immortalized cells (See, e.g., Hayflick and
Moorhead, 1971, Exp. Cell. Res., 25:585-621; and Hayflick, 1965,
Exp. Cell Research, 37:614-636, incorporated herein by reference in
their entireties, including all figures, tables and drawings.)
Therefore, an immortalized cell line can be distinguished from
non-immortalized cell lines if the cells in the cell line are able
to undergo more than 60 divisions. If the cells of a cell line are
able to undergo more than 60 cell divisions, the cell line is an
immortalized or permanent cell line. The immortalized cells of the
invention are preferably able to undergo more than 70 divisions,
are more preferably able to undergo more than 90 divisions, and are
most preferably able to undergo more than 90 cell divisions.
[0044] Typically, immortalized or permanent cells can be
distinguished from non-immortalized and non-permanent cells on the
basis that immortalized and permanent cells can be passaged at
densities lower than those of non-immortalized cells. Specifically,
immortalized cells can be grown to confluence (e.g., when a cell
monolayer spreads across an entire plate) when plating conditions
do not allow physical contact between the cells. Hence,
immortalized cells can be distinguished from non-immortalized cells
when cells are plated at cell densities where the cells do not
physically contact one another.
[0045] "Culture"--In the present invention this term refers to one
or more cells that are static or undergoing cell division in a
liquid medium. Nearly any type of cell can be placed in cell
culture conditions. Cells may be cultured in suspension and/or in
monolayers with one or more substantially similar cells. Cells may
be cultured in suspension and/or in monolayers with heterogeneous
population cells. The term heterogeneous as utilized in the
previous sentence can relate to any cell characteristics, such as
cell type and cell cycle stage, for example. Cells may be cultured
in suspension and/or in monolayers with feeder cells.
[0046] "Feeder Cells"--This refers to cells grown in co-culture
with other cells. Feeder cells include, e.g., fibroblasts, fetal
cells, oviductal cells, and may provide a source of peptides,
polypeptides, electrical signals, organic molecules (e.g.,
steroids), nucleic acid molecules, growth factors, cytokines, and
metabolic nutrients to cells co-cultured therewith. Some cells
require feeder cells to be grown in tissue culture.
[0047] "Reprogram"--This term as used in the present invention
refers to materials and methods that can convert a differentiated
cell into a less differentiated, more primitive cell type, e.g., an
embryonic stem cell.
[0048] "Embryo"--In the present invention this refers to a
developing cell mass that has not implanted into the uterine
membrane of a maternal host. Hence, the term "embryo" as used
herein can refer to a fertilized oocyte, a cybrid (defined herein),
a pre-blastocyst stage developing cell mass, and/or any other
developing cell mass that is at a stage of development prior to
implantation into the uterine membrane of a maternal host. Embryos
of the invention may not display a genital ridge. Hence, an
"embryonic cell" is isolated from and/or has arisen from an
embryo.
[0049] "Fetus"--In the present invention refers to a developing
cell mass that has implanted into the uterine membrane of a
maternal host. A fetus can include such defining features as a
genital ridge, for example. A genital ridge is a feature easily
identified by a person of ordinary skill in the art and is a
recognizable feature in fetuses of most animal species.
[0050] "Fetal Cell"--as used herein can refer to any cell isolated
from and/or has arisen from a fetus or derived from a fetus,
[0051] "Non-Fetal Cell"--refers to a cell that is not derived or
isolated from a fetus.
[0052] "Senescence"--In the present invention this refers to the
characteristic slowing of growth of non-immortal somatic cells in
tissue culture after cells have been maintained in culture for a
prolonged period. Non-immortal cells characteristically have a
defined life-span before they become senescent and die. The present
invention alleviates or prevents senescence by the introduction of
cytoplasm from a donor cell, typically an oocyte or blastomere,
into a recipient cell, e.g., a cultured human somatic cell.
DETAILED DESCRIPTION OF THE INVENTION
[0053] As explained above, the present invention provides novel
methods for de-differentiating and/or altering the life-span of
desired cells, preferably mammalian cells and, most preferably,
human or other primate cells by the introduction of cytoplasm from
a more primitive cell type, typically an undifferentiated or
substantially undifferentiated cell, e.g., an oocyte or
blastomere.
[0054] As noted previously, it was recently reported in the popular
press that a group working in the area of artificial insemination
and infertility successfully transferred the cytoplasm from the
oocyte of a younger woman into that of an older woman and thereby
rejuvenated the ability of the older oocyte to be competent for
fertilization and embryo development. Based on this anecdotal
evidence, coupled with recent papers in the scientific literature
which suggest that differentiated adult cells may be effectively
"reprogrammed" by nuclear transfer, it was theorized that
differentiated cells could be effectively "reprogrammed" or
"de-differentiated" and/or have their life-span altered (increased)
by the introduction of cytoplasm from that of undifferentiated or
substantially undifferentiated cell, e.g., an oocyte or blastomere
or another embryonic cell type.
[0055] While it is presently unknown how the cytoplasm of one cell
affects the life-span or state of differentiation of another, it is
theorized that the cytoplasm of cells in early or primitive states
of development contains one or more substances, e.g., transcription
factors and/or other substances that act to trigger or promote cell
differentiation. For example, one substance likely contained
therein that affects the state of cell differentiation is
telomerase. Another substance is OCT-4 and REX. However, Applicant
does not wish to be bound to this theory as it is not necessary for
an understanding of the invention.
[0056] In the present invention, a recipient cell will typically be
dedifferentiated in vitro by the introduction of an effective
amount of cytoplasm from a donor cell, i.e., an undifferentiated or
substantially undifferentiated cell, e.g., an oocyte or blastomere.
This introduction or transfer of cytoplasm can be effected by
different methods, e.g., by microinjection or by use of a liposomal
delivery system. A preferred means comprises the introduction of
cytoplasm blebs derived from ES cells, oocytes or other embryonic
cells into desired differentiated cells, e.g., mammalian or other
cells which are at or near senescence. For example, such cytoplasm
blebs can be introduced into genetically modified mammalian cells
in order to rejuvenate such cells, e.g., prior to their usage for
cell therapy.
[0057] Alternatively, cytoplasmic blebs can be contacted with
nuclei from differentiated cells to induce rejuvenation.
[0058] The recipient cell can be of any species and may be
heterologous to the donor cell, e.g., amphibian, mammalian, avian,
with mammalian cells being preferred. Especially preferred
recipient cells include human and other primate cells, e.g.,
chimpanzee, cynomolgus monkey, baboon, other Old World monkey
cells, caprine, equine, porcine, ovine, and other ungulates,
murine, canine, feline, and other mammalian species.
[0059] Also, the recipient cell can be any differentiated cell
type. Suitable examples thereof include epithelial cells,
endothelial cells, fibroblasts, keratinocytes, melanocytes and
other skin cell types, muscle cells, bone cells, immune cells such
as T and B-lymphocytes, oligodendrocytes, dendritic cells,
erythrocytes and other blood cells; pancreatic cells, neural and
nerve cell types, stomach, intestinal, esophageal, lung, liver,
spleen, kidney, bladder, cardiac, thymus, corneal, and other ocular
cell types, etc. In general, the methods have application in any
application wherein a source of cells that are in a less
differentiated state would be desirable.
[0060] As noted, the transferred cytoplasm will be obtained from a
"donor" cell that is in a less differentiated state or more
primitive state than the recipient cell. Typically, the cytoplasm
will be derived from oocytes or cells of early stage embryos, e.g.,
blastomeres or inner cell mass cells derived from early stage
embryos, in general, it is preferred that the donor cytoplasm be
obtained from oocytes or other embryonic cells that are in an
undifferentiated or substantially undifferentiated state. Bovine
oocytes are a preferred source because they can be readily obtained
in large quantities from slaughterhouses.
[0061] Recently there have been reports in the literature
concerning the production of cultures comprising embryonic stem
cells that reportedly express or do not express certain markers
characteristic of embryonic stem cells. It is therefore also
preferable that donor cytoplasm be obtained from an oocyte or other
cell that expresses or does not express cell markers which are
characteristic of an undifferentiated, embryonic cell type. Such
markers on primate ES cells include, by way of example, SSEA-1 (-);
SSEA-3 (+); SSEA-4 (+); TRA-1-60 (+); TRA-1-81 (+); and alkaline
phosphatase (+). (See U.S. Pat. No. 5,843,780 to Thomson, issued
Dec. 1, 1998.)
[0062] As discussed above, it is also desirable that telomerase
and/or a DNA sequence or other compound that provides for the
expression of telomerase be introduced into the recipient cell,
e.g., a mammalian cell and, more preferably, a human or non-human
primate cell. The isolation of telomerase and cloning of the
corresponding DNA has been reported prior to the present invention.
For example, WO 98/14593, published Apr. 9, 1998, by Cech et al,
reports telomerase nucleic acid sequences derived from Eeuplotes
aediculatus, Saccharomyces, Schizosaccharomyces, and human, as well
as polypeptides comprising telomerase protein subunits. Also, WO
98/14592, to Cech et al, published Apr. 9, 1998, discloses
compositions containing human telomerase reverse transcriptase, the
catalytic protein subunit of human telomerase. Also, U.S. Pat. Nos.
5,837,857 and 5,583,414 describe nucleic acids encoding mammalian
telomerases.
[0063] Still further, U.S. Pat. No. 5,830,644, issued to West et
al; U.S. Pat. No. 5,834,193, issued to Kzolowski et al, and U.S.
Pat. No. 5,837,453, issued to Harley et al, describe assays for
measuring telomerase length and telomerase activity and agents that
affect telomerase activity. These patents and PCT applications are
incorporated by reference in their entirety herein.
[0064] Thus, in the present invention, desired cells, e.g.,
cultured human somatic cells, may be de-differentiated or
reprogrammed in tissue culture by the introduction of cytoplasm of
a more primitive cell type, e.g., an oocyte or embryonic cell type
alone or in conjunction with telomerase. The introduction of
cytoplasm from a donor oocyte or embryonic cell, e.g., blastomere,
may be accomplished by various methods. For example, this can be
effected by microsurgically removing part or all of the cytoplasm
of a donor oocyte or blastomere or other embryonic cell type with a
micropipette and microinjecting such cytoplasm into that of a
recipient mammalian cell. It may also be desirable to remove
cytoplasm from the recipient cell prior to such introduction. Such
removal may be accomplished by well-known microsurgical methods.
Alternatively, the cytoplasm and/or telomerase or telomerase DNA
can be introduced using a liposomal delivery system.
[0065] The present methods should provide a means of producing
embryonic stem cells, e.g., mammalian embryonic stem cells, and
most desirably, human embryonic stem cells, by reprogramming or
de-differentiating desired cells in tissue culture. These cells are
desirable from a therapeutic standpoint since such cells can be
used to give rise to any differentiated cell type. The resultant
differentiated cell types may be used in cell transplantation
therapies.
[0066] Another significant application of the present invention is
for gene therapy. To date, many different genes of significant
therapeutic importance have been identified and cloned. Moreover,
methods for stably introducing such DNAs into desired cells, e.g.,
mammalian cells and, more preferably, human somatic cell types, are
well known. Also, methods for effecting site-specific insertion of
desired DNAs via homologous recombination are well known in the
art.
[0067] However, while suitable vectors and methods for introduction
and detection of specific DNAs into desired somatic cells are
known, a significant obstacle to the efficacy of such methods is
the limited life-span of normal, i.e., non-immortal cells, in
tissue culture. This is particularly problematic in situations
wherein the introduction of multiple DNA modifications, e.g.,
deletions, substitutions, and/or additions is desired. Essentially,
while methods for effecting targeted DNA modifications are known,
the requisite time to effect and select for such modifications can
be very lengthy. Thus, the cells may become senescent or die before
the desired DNA modifications have been effected.
[0068] The present invention will alleviate this inherent
constraint of gene and cell therapy by introducing the cytoplasm of
an oocyte or other embryonic cell type into recipient cells prior,
concurrent or subsequent to genetic modification. The introduction
of such cytoplasm alone or in combination with telomerase or a DNA
or another compound that results in the expression of telomerase,
will reprogram the genetically modified cell and enable it to have
a longer life-span in tissue culture. Such reprogramming can be
effected once or repeatedly during genetic modification of
recipient cells. For example, in the case of very complex genetic
modifications, it may be necessary to "reprogram" recipient cells
several times by the repeated introduction of donor cytoplasm to
prevent senescence. The optimal frequency of such reprogramming
will be determined by monitoring the doubling time of the cells in
tissue culture such that the cells are reprogrammed before they
become senescent.
[0069] The resultant reprogrammed genetically modified cells, which
have a longer life-span as a result of reprogramming, may be used
for cell and gene therapy. Moreover, these cells may be used as
donor cells for nuclear transfer procedures or for the production
of chimeric animals. The present methods will make it possible to
produce cloned and chimeric animals having complex genetic
modifications. This will be especially advantageous for the
production of animal models for human diseases. Also, the present
methods will be beneficial in situations wherein the expression of
a desired gene product or phenotype is dependent upon the
expression of different DNA sequences, or for gene research
involving the interrelated effects of different genes on one
another. Moreover, it is anticipated that the present methods will
become very important as the interrelated effects of the expression
of different genes on others becomes more understood.
[0070] Yet another application of the present invention is for
alleviating the effects of aging. Just as mammalian cells have a
finite life-span in tissue culture, they similarly have a finite
life-span in vivo. This finite life-span is hypothesized to explain
why organisms, including humans, have a normal maximum life-span,
determined by the finite life-span of human somatic cells.
[0071] The present invention will alleviate the effects of aging by
taking mammalian cells from an individual and altering
(lengthening) the life-span of such cells by introduction of
cytoplasm from an oocyte or other embryonic cell type, e.g.,
blastomere. The resultant rejuvenated cells may be used to produce
differentiated cell types in tissue culture and these cells can
then be introduced into the individual. This can be used, e.g., to
rejuvenate the immune system of an individual. Such rejuvenation
should be useful in the treatment of diseases thought to be of
immune origin, e.g., some cancers.
[0072] Also, the subject methods may be used for the production of
autologous grafts, e.g., skin grafts, which can be used in the case
of tissue injury or elective surgery.
[0073] Yet another application of the present application is for
treating the effects of chronologic and UV-induced aging on the
skin. As skin ages, various physical changes may be manifested
including discoloration, loss of elasticity, loss of radiance, and
the appearance of fine lines and wrinkles. It is anticipated that
such effects of aging may be alleviated or even reversed by topical
application of cytoplasm-containing compositions. For example,
cytoplasm from donor oocytes, e.g., bovine oocytes, optionally
further including telomerase or a telomerase DNA construct, can be
packaged in liposomes to facilitate internalization into skin cells
upon topical application. Also, it may be advantageous to include
in such compositions compounds that facilitate absorption into the
skin, e.g., DMSO. These compositions may be topically applied to
areas of the skin wherein the effects of aging are most pronounced,
e.g., the skin around the eyes, the neck and the hands.
[0074] Still another application of the present invention is for
identification of the substance or substances found in cytoplasm
that induces de-differentiation. This can be effected by
fractionation of cytoplasm and screening these fractions to
identify those which contain substances that result in effective
rejuvenation or reprogramming when transferred into recipient
cells, e.g., human differentiated cell types.
[0075] Alternatively, the components) contained in oocyte cytoplasm
responsible for reprogramming or rejuvenation can be identified by
sub tractive hybridization by comparing mRNA expression in early
stage embryos and oocytes to that of more differentiated
embryos.
[0076] With respect to such identification, it is currently unknown
what component or compounds contained in embryonic cell cytoplasm
are responsible for cell reprogramming or de-differentiation. In
fact, it is uncertain even as to the specific nature of such
component(s), e.g., whether they are nucleic acids or
proteinases.
[0077] However, it is speculated by the present inventors that such
component(s) may comprise nucleic acids, in particular maternal
RNAs, or proteins encoded thereby. In this regard, it has been
reported by different groups that very early stage embryos contain
a class of RNA known as maternal RNA's that are stored in the egg
very early on but which are not detected past the blastula stage.
(Kontrogianni-Konstantopoulos et al, Devel. Biol., 177(2):371-382
(1996).) Maternal RNA levels have been quantified for different
species, i.e., rabbit, cow, pig, sheep and mouse. (Olszanska et al,
J. Exp. Zool., 265(3):317-320 (1993).) With respect thereto, it has
also been reported that maternal RNA in Drosophila oocyte encodes a
protein that may bind to a tyrosine kinase receptor present in
adjacent follicle cells that may initiate various events leading to
dorsal follicle cell differentiation which act to delimit and
orient the future dorsoventral axis of the embryo. (Schupbach et
al, Curr. Opin. Genet. Dev., 4(4):502-507 (1994).)
[0078] Also, fractionation of oocytes has shown that
mitogen-activated protein kinases are expressed at higher levels in
small oocytes, suggesting that it is a maternal RNA that is stored
for early embryogenesis. This is speculated to be involved in
signal transduction in embryonic as well as adult cells.
(Zaitsevskaya et al, Cell Growth Differ., 3(11):773-782
(1992).)
[0079] Still further, it has been reported that a maternal mRNA in
silkworm oocytes encodes a protein that may be a structural
component necessary for formation of the cellular blastoderm of the
embryo, and that the association of such maternal mRNA with
cortical cytoskeleton may participate in the synthesis of new
cytoskeleton or related structures during blastoderm development.
(Kastern et al, Devel., 108(3):497-505(1990).)
[0080] Moreover, it has been reported that maternal poly(A)+ RNA
molecules found in the egg of the sea urchin and amphibian oocyte
are completed with U1 RNA, a co-factor in somatic nuclear pre-mRNA
splicing and that such RNAs contain repeated sequences interspersed
with single-copy elements. (Calzone et al, Genes Devel.,
2(3):305-318 (1988); Ruzdijic et al, Development, 101(1):107-116
(1987).)
[0081] Thus, based thereon, and the observation that cytoplasm
apparently contains some component that results in cell
reprogramming, it should be possible to identify compounds, likely
nucleic acids and/or proteinaceous compounds which are present in
the cytoplasm of oocytes and early embryos that, under appropriate
conditions, provide for reprogramming or de-differentiation of
desired cells. This will be effected by fractionation of cytoplasm
into different fractions, e.g., based on size or isoelectric point,
and ascertaining those factors which effect de-differentiation or
reprogramming when transferred to differentiated cell types.
[0082] Alternatively, the factors responsible for reprogramming may
be identified by sub tractive or differential hybridization,
essentially by identifying those mRNAs which are present in oocytes
that are lost after the embryo has differentiated beyond a certain
stage, e.g., past the blastula stage of development, and
identifying those of which are involved in de-differentiation or
reprogramming.
[0083] Therefore, the invention includes the identification of the
specific cytoplasmic materials, e.g., polypeptides and/or nucleic
acid sequences, which when transferred into a differentiated cell
provide for de-differentiation or reprogramming. Based on what has
been reported with respect to maternal RNAs, it is anticipated that
the active materials responsible for de-differentiation or
reprogramming may include maternal RNAs or polypeptides encoded
thereby.
[0084] After such nucleic acid(s) or polypeptides have been
identified and sequenced, they will be produced by recombinant
methods. It is anticipated that these recombinantly produced
nucleic acids or polypeptides will be sufficient to induce
reprogramming or de-differentiation of desired cells.
[0085] The invention further encompasses assays wherein oocyte
cytomplasm or cytoplasm from ES cells is fractionated into
different fractions, e.g., based on molecular weight, isoelectric
point, gel filtration, and salt precipitation, which are added into
different microwells that contain one or more isolated nuclei from
desired differentiated cells, e.g., mammalian, amphibian, avian, or
insect cells and a screening assay conducted to identify mRNAs such
as REX or OCT-4 that are released from the nuclei. For example,
such mRNAs may be identified by PCR amplification and
detection.
[0086] Alternatively, PCR screening assays may be conducted'
wherein ooplasm can be added to desired differentiated cells and
assays conducted to identify what mRNAs, e.g., REX or OCT-4, are
released from the cell nuclei after introduction of the oocyte
cytoplasm.
[0087] The identification of such mRNAs can be identified by known
methods, e.g., subtractive hybridization, differential display, and
differential hyridization techniques. Essentially, these methods
provide for the comparison of different populations of mRNAs in
different cells, or cells at different times, and are
conventionally used to identify genes that are expressed only under
specific conditions or by specific types of cells.
[0088] In particular, subtractive hybridization can be effected by
use of oocyte RNAs which are subtracted with RNAs obtained from
normal somatic cell RNAs. Thereby, RNAs that are involved in cell
reprogramming can be identified.
[0089] Additionally, the invention further includes the
reconstitution of nuclei isolated from desired differentiated
cells, e.g., those which are derived from differentiated cells in
tissue culture, which potentially may be genetically modified by
contacting such isolated nuclei with cytoplasm fractionated from
oocytes, blastomeres or ES cells, and the addition of such
reconstituted nuclei to cytoplasts, thereby producing a rejuvenated
cell having increased proliferation potential and lifespan.
* * * * *