U.S. patent application number 12/835026 was filed with the patent office on 2012-10-04 for porcine animals lacking any expression of functional alpha 1,3 galactosyltransferase.
Invention is credited to David L. Ayares, Carol J. Phelps.
Application Number | 20120255047 12/835026 |
Document ID | / |
Family ID | 32043169 |
Filed Date | 2012-10-04 |
United States Patent
Application |
20120255047 |
Kind Code |
A1 |
Phelps; Carol J. ; et
al. |
October 4, 2012 |
Porcine Animals Lacking Any Expression of Functional Alpha 1,3
Galactosyltransferase
Abstract
The present invention is a porcine animal, tissue, organ, cells
and cell lines, which lack any expression of functional alpha 1,3
galactosyltransferase (alpha1,3GT). These animals, tissues, organs
and cells can be used in xenotransplantation and for other medical
purposes.
Inventors: |
Phelps; Carol J.; (Floyd,
VA) ; Ayares; David L.; (Blacksburg, VA) |
Family ID: |
32043169 |
Appl. No.: |
12/835026 |
Filed: |
July 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10646970 |
Aug 21, 2003 |
7795493 |
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12835026 |
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60404775 |
Aug 21, 2002 |
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Current U.S.
Class: |
800/17 ; 435/325;
800/21; 800/22 |
Current CPC
Class: |
A01K 2267/02 20130101;
A01K 2267/025 20130101; C12N 15/8509 20130101; C12N 9/2465
20130101; A01K 2227/108 20130101; A01K 2217/075 20130101; A01K
67/0276 20130101; A61P 37/06 20180101 |
Class at
Publication: |
800/17 ; 435/325;
800/21; 800/22 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12N 15/09 20060101 C12N015/09; C12N 5/10 20060101
C12N005/10 |
Claims
1. A pig that lacks any expression of functional alpha1,3
galactosyltransferase.
2. An organ of a pig that lacks any expression of functional
alpha1,3 galactosyltransferase.
3. The organ of claim 2, wherein the organ is a kidney.
4. The organ of claim 2, wherein the organ is a liver.
5. The organ of claim 2, wherein the organ is a heart.
6. The organ of claim 2, wherein the organ is a lung.
7. The organ of claim 2, wherein the organ is a pancreas.
8. A tissue of a pig that lacks any expression of functional
alpha1,3 galactosyltransferase.
9. The tissue of claim 8, wherein the tissue is cartilage.
10. The tissue of claim 8, wherein the tissue is bone.
11. The tissue of claim 8, wherein the tissue is adipose.
12. The tissue of claim 8, wherein the tissue is muscle.
13. A cell or a cell line from a pig that lacks any expression of
functional alpha1,3 galactosyltransferase.
14. The cell of claim 13, wherein the cell is derived from the
pancreas.
15. The cell of claim 14, wherein the cell is an Islet of
Langerhans cell.
16. The cell of claim 14, wherein the cell is an insulin secreting
cell.
17. A method for the production of a pig that lacks any expression
of functional alpha1,3 galactosyltransferase comprising: breeding a
male pig heterozygous for the alpha-1,3-GT gene with a female pig
heterozygous for the alpha-1,3-GT gene.
18. The method of claim 17, wherein one or both pigs are
heterozygous due to the genetic modification of one allele of the
alpha-1,3-GT gene to prevent expression of that allele.
19. The method of claim 17, wherein one or both pigs are
heterozygous due to the presence of a point mutation in an allele
of the alpha-1,3-GT gene.
20. The method of claim 19, wherein the point mutation is a T-to-G
point mutation at the second base of exon 9 of the alpha-1,3-GT
gene.
21. The method of claim 17, wherein a male pig that contains a
T-to-G point mutation at the second base of exon 9 of the
alpha-1,3-GT gene is bred with a female pig that contains a T-to-G
point mutation at the second base of exon 9 of the alpha-1,3-GT
gene.
22. A method for producing an alpha 1,3 GT deficient non-human
animal comprising: (a) exposing a population of cells to C.
difficile toxin A; (b) removing cells which lift from the surface
matrix because they are adversely affected by toxin A due to the
receptor-mediated cytotoxicity of the toxin; (c) expanding and
maintaining those cells which do not show the effects of
receptor-mediated cytotoxicity; (d) using these toxin A-resistant
cells as nuclear donors for nuclear transplantation into a suitable
recipient cell; (e) implanting the fused and activated cells into a
female surrogate; and (f) producing a cloned animal which exhibits
a deficiency or complete lack of gal alpha1,3-gal epitopes on its
cell surfaces.
23. The method of claim 22, wherein the cells to be selected for
have been rendered heterozygous with respect to the gal alpha1,3
allele, via targeted knockout of one allele by homologous
recombination.
24. The method of claim 22, wherein the cells to be selected for
have been rendered homozygous with respect to the gal alpha1,3
allele, via targeted knockout of both alleles by homologous
recombination.
25. The method of claim 22 wherein the cells to be selected for
have been rendered heterozygous with respect to the gal alpha1,3
allele via a natural mutation of a single gal alpha1,3 allele,
which disables the alpha 1,3 galactosyltransferase gene.
26. The method of claim 22 wherein the cells to be selected carry
an alpha 1,3 gal double knockout created by targeted knockout of
one allele by homologous recombination and natural mutation of the
second allele.
27. The method of claim 22 wherein the cells to be selected for are
homozygous with respect to the gal alpha1,3 allele accomplished via
natural mutations of both gal alpha1,3 alleles, which disables the
alpha 1,3 galactosyltransferase gene.
28. The method of claim 22 wherein the cells to be selected for
carry an alpha 1,3 gal double knockout accomplished via targeted
knockout of one allele by homologous recombination and natural
mutation of the second allele which disables the alpha 1,3
galactosyltransferase gene.
29. The method of claim 22, wherein the cells to be selected for
have been rendered homozygous with respect to the gal alpha1,3
allele via induced mutations of both gal alpha1,3 alleles, which
disables the alpha 1,3 galactosyltransferase gene.
30. The method of claim 22 wherein the Clostridium difficile toxin
A used for selection is in the form of a purified toxin.
31. The method of claim 22 wherein the Clostridium difficile toxin
A used for selection is in the form of a culture supernatant
fluid.
32. The method of claim 22 wherein the purified toxin is applied to
dispersed cells, and wherein said dispersed cells are then cultured
in vitro in vessels suitable for cell adherence.
33. The method of claim 22, wherein the purified toxin is applied
to adhered cells.
34. The method of claim 22, wherein the culture supernatant fluid
is applied to dispersed and un-adhered cells followed by culturing
in vessels suitable for cell adherence.
35. The method of claim 22, wherein the culture supernatant fluid
is applied
36. The method of claim 22, wherein said animal is a pig.
37. The method of claim 22, wherein a mutation is induced by a
mutagenic agent selected from the group consisting of a chemical
mutagen, radiation, and a transposon.
38. The method of claim 22, wherein said chemical mutagen is
selected from the group consisting of EMS, ENU, mustard gas and
ICR191.
39. The method of claim 22, wherein said radiation is selected from
the group consisting of ultraviolet radiation, alpha radiation,
beta radiation and gamma radiation.
40. A cell that carries a homozygous knockout for the gal
alpha-1,3-GT gene in which at least one allele contains a natural
or spontaneous mutation in the gal alpha-1,3-GT gene, wherein said
cell is produced by a method comprising: (a) exposing a population
of cells to C. difficile toxin A; (b) removing cells which are
adversely affected by toxin A due to the receptor-mediated
cytotoxicity of the toxin; and (c) expanding and maintaining a cell
that does not show the effects of receptor-mediated
cytotoxicity.
41. The cell of claim 40, wherein said cell carries a homozygous
knockout for the gal alpha-1,3-GT gene in which at least one allele
contains the base substitution thymine to guanine at base position
424 of the alpha 1,3 GT gene, resulting in the amino acid
substitution tyrosine to aspartic acid at position 142 in the gal
alpha-1,3-GT protein.
42. The cell of claim 40, wherein said cell carries a homozygous
knockout for the gal alpha-1,3-GT gene in which at least one allele
contains an induced mutation in the gal alpha-1,3-GT gene.
43. An animal produced according to the method of claim 22.
44. An animal produced by nuclear transfer cloning using the cell
of claim 40 as a nuclear donor.
45. An animal produced by nuclear transfer cloning using the cell
of claim 41 as a nuclear donor.
46. An animal produced by nuclear transfer cloning using the cell
of claim 42 as a nuclear donor.
47. Cells, tissues, and organs obtained from the animal of claim 42
for use as an in vivo or ex vivo supplement or replacement for
recipient cells, tissues or organs.
48. Cells, tissues, and organs obtained from the animal of claim 43
for use as an in vivo or ex vivo supplement or replacement for
recipient cells, tissues, or organs.
49. Cells, tissues, and organs obtained from the animal of claim 44
for use as an in vivo or ex vivo supplement or replacement for
recipient cells, tissues, or organs.
50. Cells, tissues, and organs obtained from the animal of claim 45
for use as an in vivo or ex vivo supplement or replacement for
recipient cells, tissues, or organs.
Description
[0001] This application claims priority to U.S. provisional patent
application No. 60/404,775 filed Aug. 21, 2002.
FIELD OF THE INVENTION
[0002] The present invention are porcine animals, tissue and organs
as well as cells and cell lines derived from such animals, tissue
and organs, which lack any expression of functional alpha 1,3
galactosyltransferase (alpha1,3GT). Such animals, tissues, organs
and cells can be used in research and in medical therapy, including
in xenotransplantation.
BACKGROUND OF THE INVENTION
[0003] Patients with end stage organ failure require organ
transplantation for survival. The major limiting factor in clinical
transplantation is the shortage of suitable human donors. Over the
past ten years the size of the waiting list of patients for organs
has increased dramatically, from approximately 30,000 in 1991 to
approximately 80,000 in 2001 (Source: New York Organ Donor
Network.; Association of Organ Procurement Organizations' Death
Record Review Study from 1997 to 1999, provided by 30 organ
procurement organizations). Despite this increasing need over the
past ten years, the number of organ donations has remained flat
(approximately 20,000 per year).
[0004] According to the United Network for Organ Sharing (UNOS) as
of Jul. 17, 2003, there were 82,249 patients waiting for organ
transplants in the United States. The need for specific organs was
as follows:
TABLE-US-00001 Kidney 55,133 Liver 17,304 Pancreas 1,413 Kidney and
Pancreas 2,378 Intestine 173 Heart 3,717 Heart-Lung 184 Lung
3,912
[0005] Across the U.S., an average of 17 men, women and children of
all races and ethnic backgrounds die every day for lack of donated
organs, thus, each year, more than 6,200 Americans die waiting for
an organ transplant. A need for a more reliable and unlimited
source of organs has led to investigation of the potential for
transplantation of organs from other animals, referred to as
xenotransplantation.
[0006] Pigs are considered the most likely source of xenograft
organs. The supply of pigs is plentiful, breeding programs are well
established, and their size and physiology are compatible with
humans. Xenotransplantation, however, presents its own set of
problems. The most significant is immune rejection. The first
immunological hurdle is "hyperacute rejection" (HAR). HAR can be
defined by the ubiquitous presence of high titers of pre-formed
natural antibodies binding to the foreign tissue. The binding of
these natural antibodies to target epitopes on the donor organ
endothelium is believed to be the initiating event in HAR. This
binding, within minutes of perfusion of the donor organ with the
recipient blood, is followed by complement activation, platelet and
fibrin deposition, and ultimately by interstitial edema and
hemorrhage in the donor organ, all of which cause failure of the
organ in the recipient (Strahan et al. (1996) Frontiers in
Bioscience 1, e34-41).
[0007] Except for Old World monkeys, apes and humans, most mammals
carry glycoproteins on their cell surfaces that contain galactose
alpha 1,3-galactose (Galili et al., J. Biol. Chem. 263:
17755-17762, 1988). Humans, apes and Old World monkeys have a
naturally occurring anti-alpha gal antibody that is produced in
high quantity (Cooper et al., Lancet 342:682-683, 1993). It binds
specifically to glycoproteins and glycolipids bearing galactose
alpha-1,3 galactose.
[0008] In contrast, glycoproteins that contain galactose alpha
1,3-galactose are found in large amounts on cells of other mammals,
such as pigs. This differential distribution of the "alpha-1,3 GT
epitope" and anti-Gal antibodies (i.e., antibodies binding to
glycoproteins and glycolipids bearing galactose alpha-1,3
galactose) in mammals is the result of an evolutionary process
which selected for species with inactivated (i.e. mutated)
alpha-1,3-galactosyltransferase in ancestral Old World primates and
humans. Thus, humans are "natural knockouts" of alpha1,3GT. A
direct outcome of this event is the rejection of xenografts, such
as the rejection of pig organs transplanted into humans initially
via HAR.
[0009] A variety of strategies have been implemented to eliminate
or modulate the anti-Gal humoral response caused by porcine
xenotransplantation, including enzymatic removal of the epitope
with alpha-galactosidases (Stone et al., Transplantation 63:
640-645, 1997), specific anti-gal antibody removal (Ye et al.,
Transplantation 58: 330-337, 1994), capping of the epitope with
other carbohydrate moieties, which failed to eliminate alpha-1,3-GT
expression (Tanemura et al., J. Biol. Chem. 27321: 16421-16425,
1998 and Koike et al., Xenotransplantation 4: 147-153, 1997) and
the introduction of complement inhibitory proteins (Dalmasso et
al., Clin. Exp. Immunol. 86: 31-35, 1991, Dalmasso et al.
Transplantation 52:530-533 (1991)). C. Costa et al. (FASEB J 13,
1762 (1999)) reported that competitive inhibition of alpha-1,3-GT
in H-transferase transgenic pigs results in only partial reduction
in epitope numbers. Similarly, S. Miyagawa et al. (J Biol. Chem.
276, 39310 (2001)) reported that attempts to block expression of
gal epitopes in N-acetylglucosaminyltransferase III transgenic pigs
also resulted in only partial reduction of gal epitopes numbers and
failed to significantly extend graft survival in primate
recipients.
[0010] Single allele knockouts of the alpha-1,3-GT locus in porcine
cells and live animals have been reported. Denning et al. (Nature
Biotechnology 19: 559-562, 2001) reported the targeted gene
deletion of one allele of the alpha-1,3-GT gene in sheep. Harrison
et al. (Transgenics Research 11: 143-150, 2002) reported the
production of heterozygous alpha-1,3-GT knock out somatic porcine
fetal fibroblasts cells. In 2002, Lai et al. (Science 295:
1089-1092, 2002) and Dai et al. (Nature Biotechnology 20: 251-255,
2002) reported the production of pigs, in which one allele of the
alpha-1,3-GT gene was successfully rendered inactive. Ramsoondar et
al. (Biol of Reproduc 69, 437-445 (2003) reported the generation of
heterozygous alpha-1,3-GT knockout pigs that also express human
alpha-1,2-fucosyltransferase (HT), which expressed both the HT and
alpha-1,3-GT epitopes.
[0011] PCT publication No. WO 94/21799 and U.S. Pat. No. 5,821,117
to the Austin Research Institute; PCT publication No. WO 95/20661
to Bresatec; and PCT publication No. WO 95/28412, U.S. Pat. No.
6,153,428, U.S. Pat. No. 6,413,769 and US publication No.
2003/0014770 to BioTransplant, Inc. and The General Hospital
Corporation provide a discussion of the production of alpha-1,3-GT
negative porcine cells based on knowledge of the cDNA of the
alpha-1,3-GT gene (and without knowledge of the genomic
organization or sequence). However, there was no evidence that such
cells were actually produced prior to the filing date of these
applications and the Examples were all prophetic.
[0012] The first public disclosure of the successful production of
a heterozygous alpha-1,3-GT negative porcine cell occurred in July
1999 at the Lake Tahoe Transgenic Animal Conference (David Ayares,
et al., PPL Therapeutics, Inc.). Prior to the present invention, no
one had published or publicly disclosed the production of a
homozygous alpha 1,3GT negative porcine cell. Further, since
porcine embryonic stem cells have not been available to date, there
was and still is no way to use an alpha-1,3-GT homogygous embryonic
stem cell to attempt to prepare a live homogygous alpha-1,3-GT
knock out pig.
[0013] On Feb. 27, 2003, Sharma et al. (Transplantation 75:430-436
(2003) published a report demonstrating a successful production of
fetal pig fibroblast cells homozygous for the knockout of the
alpha-1,3-GT gene.
[0014] PCT publication No. WO 00/51424 to PPL Therapeutics
describes the genetic modification of somatic cells for nuclear
transfer. This patent application discloses the genetic disruption
of the alpha-1,3-GT gene in porcine somatic cells, and the
subsequent use of the nucleus of these cells lacking at least one
copy of the alpha-1,3-GT gene for nuclear transfer.
[0015] U.S. Pat. No. 6,331,658 to Cooper & Koren claims but
does not confirm any actual production of genetically engineered
mammals that express a sialyltransferase or a fucosyltransferase
protein. The patent asserts that the genetically engineered mammals
would exhibit a reduction of galactosylated protein epitopes on the
cell surface of the mammal.
[0016] PCT publication No. WO 03/055302 to The Curators of the
University of Missouri confirms the production of heterozygous
alpha 1,3GT knockout miniature swine for use in
xenotransplantation. This application is generally directed to a
knockout swine that includes a disrupted alpha-1,3-GT gene, wherein
expression of functional alpha-1,3-GT in the knockout swine is
decreased as compared to the wildtype. This application does not
provide any guidance as to what extent the alpha-1,3-GT must be
decreased such that the swine is useful for xenotransplantation.
Further, this application does not provide any proof that the
heterozygous pigs that were produced exhibited a decreased
expression of functional alpha1,3GT. Further, while the application
refers to homozygous alpha 1,3GT knockout swine, there is no
evidence in the application that any were actually produced or
producible, much less whether the resultant offspring would be
viable or phenotypically useful for xenotransplantation.
[0017] Total depletion of the glycoproteins that contain galactose
alpha 1,3-galactose is clearly the best approach for the production
of porcine animals for xenotransplantation. It is theoretically
possible that double knockouts, or the disruption of both copies of
the alpha 1,3GT gene, could be produced by two methods: 1) breeding
of two single allele knockout animals to produce progeny, in which
case, one would predict based on Mendelian genetics that one in
four should be double knockouts or 2) genetic modification of the
second allele in a cell with a pre-existing single knockout. In
fact, this has been quite difficult as illustrated by the fact that
while the first patent application on knock-out porcine cells was
filed in 1993, the first homozygous alpha 1,3GT knock out pig was
not produced until July 2002 (which was based on the work of the
present inventor and described herein).
[0018] Transgenic mice (not pigs) have historically been the
preferred model to study the effects of genetic modifications on
mammalian physiology, for a number of reasons, not the least of
which is that mouse embryonic stem cells have been available while
porcine embryonic stem cells have not been available. Mice are
ideal animals for basic research applications because they are
relatively easy to handle, they reproduce rapidly, and they can be
genetically manipulated at the molecular level. Scientists use the
mouse models to study the molecular pathologies of a variety of
genetically based diseases, from colon cancer to mental
retardation. Thousands of genetically modified mice have been
created to date. A "Mouse Knockout and Mutation Database" has been
created by BioMedNet to provide a comprehensive database of
phenotypic and genotypic information on mouse knockouts and
classical mutations (http://research.bmn.com/mkmd; Brandon et al
Current Biology 5[7]:758-765 (1995); Brandon et al Current Biology
5[8]:873-881 (1995)), this database provides information on over
3,000 unique genes, which have been targeted in the mouse genome to
date.
[0019] Based on this extensive experience with mice, it has been
learned that transgenic technology has some significant
limitations. Because of developmental defects, many genetically
modified mice, especially null mice created by gene knock out
technology die as embryos before the researcher has a chance to use
the model for experimentation. Even if the mice survive, they can
develop significantly altered phenotypes, which can render them
severely disabled, deformed or debilitated (Pray, Leslie, The
Scientist 16 [13]: 34 (2002); Smith, The Scientist 14[15]:32,
(2000); Brandon et al Current Biology 5[6]:625-634 (1995); Brandon
et al Current Biology 5[7]:758-765 (1995); Brandon et al Current
Biology 5[8]:873-881 (1995); http://research.bmn.com/mkmd).
Further, it has been learned that it is not possible to predict
whether or not a given gene plays a critical role in the
development of the organism, and, thus, whether elimination of the
gene will result in a lethal or altered phenotype, until the
knockout has been successfully created and viable offspring are
produced.
[0020] Mice have been genetically modified to eliminate functional
alpha-1,3-GT expression. Double-knockout alpha-1,3-GT mice have
been produced. They are developmentally viable and have normal
organs (Thall et al. J Biol Chem 270:21437-40 (1995); Tearle et al.
Transplantation 61:13-19 (1996), see also U.S. Pat. No. 5,849,991).
However, two phenotypic abnormalities in these mice were apparent.
First, all mice develop dense cortical cataracts. Second, the
elimination of both alleles of the alpha-1,3-GT gene significantly
affected the development of the mice. The mating of mice
heterozygous for the alpha-1,3-GT gene produced genotype ratios
that deviated significantly from the predicted Mendelian 1:2:1
ratio (Tearle et al. Transplantation 61:13-19 (1996)).
[0021] Pigs have a level of cell surface glycoproteins containing
galactose alpha 1,3-galactose that is 100-1000 fold higher than
found in mice. (Sharma et al. Transplantation 75:430-436 (2003);
Galili et al. Transplantation 69:187-190 (2000)). Thus, alpha1,3-GT
activity is more critical and more abundant in the pig than the
mouse.
[0022] Despite predictions and prophetic statements, prior to this
invention, no one knew whether the disruption of both alleles of
the alpha-1,3-GT gene would be lethal or would effect porcine
development or result in an altered phenotype (Ayares et al. Graft
4(1)80-85 (2001); Sharma et al. Transplantation 75:430-436 (2003);
Porter & Dallman Transplantation 64:1227-1235 (1997); Galili,
U. Biochimie 83:557-563 (2001)). Indeed, many experts in the field
expressed serious doubts as to whether homozygous alpha-1,3-GT
knockout pigs would be viable at all, much less develop normally.
Such concerns were expressed up until the double knockout pig of
the present invention was produced. Examples of statements by those
working in the field at the time included the following.
[0023] "The abundantly expressed alpha-gal epitope may have some
biological roles in pig development, such as in cell-cell
interaction. If this assumption is correct, pigs may not develop in
the absence of this epitope (Galili, U. Biochimie 83:557-563
(2001)."
[0024] "The inability to generate knockout pigs for alpha-gal may
suggest that alpha-gal epitopes are indispensable in this species
(Galili et al. Transplantation 69:187-190 (2000))."
[0025] "Although double-knockout alpha-gal mice develop and remain
fairly normal, the possibility exists that deletion of this enzyme
could have more severe consequences in other animals (Porter &
Dallman Transplantation 64:1227-1235 (1997))."
[0026] "It is possible that the GT(-/-) pig may not be viable
because the GT gene is essential for embryonic development. An
answer to this question and to the relevance of GT(-/-) pigs to
xenotransplantation research must await, if possible, the
production of the appropriate pigs (Sharma et al. Transplantation
75:430-436 (2003))."
[0027] "Since Gal epitope expression in pig organs is up to
500-fold higher than in mouse organs, there is the possibility that
alphaGT activity is more crucial to the pig and could effect
development of these pigs (Ayares et al. Graft 4(1)80-85
(2001))."
[0028] Thus, until a viable double alpha-1,3-GT knockout pig is
produced, according to those of skill in the art at the time, it
was not possible to determine (i) whether the offspring would be
viable or (ii) whether the offspring would display a phenotype that
allows the use of the organs for transplantation into humans.
[0029] It is therefore an object of the present invention to
provide viable pigs which lack any expression of functional
alpha1,3GT.
[0030] It is another object of the present invention to provide
procine cells, tissues and organs, which lack any expression of
functional alpha1,3GT, for use in xenotransplantation or other
biomedical applications.
[0031] It is a further object of the present invention to provide a
method to select and screen for porcine cells, which lack galactose
alpha 1,3-galactose epitopes on the cell surface.
SUMMARY OF THE INVENTION
[0032] This invention is the production of the first live pigs
lacking any functional expression of alpha 1,3
galactosyltransferase. The subject of this invention was heralded
in a full paper in Science magazine in 2003 (Phelps et al. (Science
299:411-414 (2003)) and widely reported in the press as a
breakthrough in xenotransplantation.
[0033] It has for the first time been proven that a viable porcine
animal that lacks any expression of functional alpha 1,3
galactosyltransferase can be produced. The present invention
provides the complete inactivation of both alleles of the alpha 1,3
galactosyltransferase gene in pigs, thus overcoming this
longstanding hurdle and making xenotransplantation a reality.
Eliminating the expression of this gene, resulting in a lack of
galactose alpha 1,3-galactose epitopes on the cell surface,
represents the first and major step in eliminating hyperacute
rejection in pig-to-human xenotransplantation therapy. The
invention also provides organs, tissues, and cells derived from
such porcine animals, which are useful for xenotransplantation.
[0034] In embodiments of the present invention, the alleles of the
alpha-1,3-GT gene are rendered inactive, such that the resultant
alpha-1,3-GT enzyme can no longer generate galactose
alpha1,3-galactose on the cell surface. In one embodiment, the
alpha-1,3-GT gene can be transcribed into RNA, but not translated
into protein. In another embodiment, the alpha-1,3-GT gene can be
transcribed in an inactive truncated form. Such a truncated RNA may
either not be translated or can be translated into a nonfunctional
protein. In an alternative embodiment, the alpha-1,3-GT gene can be
inactivated in such a way that no transcription of the gene occurs.
In a further embodiment, the alpha-1,3-GT gene can be transcribed
and then translated into a nonfunctional protein.
[0035] In another embodiment, pigs that lack any expression of
functional alpha-1,3-GT are useful for providing a clearer
evaluation of approaches currently in development aimed at
overcoming potential delayed and chronic rejection mechanisms in
porcine xenotransplantation.
[0036] In one aspect of the present invention, porcine animals are
provided in which at least one allele of the alpha-1,3-GT gene is
inactivated via a genetic targeting event. In another aspect of the
present invention, porcine animals are provided in which both
alleles of the alpha-1,3-GT gene are inactivated via a genetic
targeting event. The gene can be targeted via homologous
recombination. In other embodiments, the gene can be disrupted,
i.e. a portion of the genetic code can be altered, thereby
affecting transcription and/or translation of that segment of the
gene. For example, disruption of a gene can occur through
substitution, deletion ("knockout") or insertion ("knockin")
techniques. Additional genes for a desired protein or regulatory
sequence that modulate transcription of an existing sequence can be
inserted.
[0037] Pigs that possess two inactive alleles of the alpha-1,3-GT
gene are not naturally occurring. The predicted frequency of
occurrence of such a pig would be in the range of 10.sup.-10 to
10.sup.-12, and has never been identified.
[0038] As one aspect of the invention, it was surprisingly
discovered that while attempting to knockout the second allele of
the alpha-1,3-GT gene through a genetic targeting event, a point
mutation was identified, which rendered the second allele inactive.
Pigs carrying point mutations in the alpha-1,3-GT gene are free of
antibiotic-resistance genes and thus have the potential to make a
safer product for human use. Thus, another aspect of the invention
is a homozygous alpha-1,3-GT knock out that has no antibiotic
resistant or other selectable marker genes. In one embodiment, this
point mutation can occur via a genetic targeting event. In another
embodiment, this point mutation can be naturally occurring. In a
further embodiment, mutations can be induced in the alpha-1,3-GT
gene via a mutagenic agent.
[0039] In one specific embodiment the point mutation can be a
T-to-G mutation at the second base of exon 9 of the alpha-1,3-GT
gene (FIG. 2). In other embodiments, at least two, at least three,
at least four, at least five, at least ten or at least twenty point
mutations can exist to render the alpha-1,3-GT gene inactive. In
other embodiments, pigs are provided in which both alleles of the
alpha-1,3-GT gene contain point mutations that prevent any
expression of functional alpha1,3GT. In a specific embodiment, pigs
are provided that contain the T-to-G mutation at the second base of
exon 9 in both alleles of the alpha-1,3-GT gene (FIG. 2).
[0040] Another aspect of the present invention provides a porcine
animal, in which both alleles of the alpha-1,3-GT gene are
inactivated, whereby one allele is inactivated by a genetic
targeting event and the other allele is inactivated via a point
mutation. In one embodiment, a porcine animal is provided, in which
both alleles of the alpha-1,3-GT gene are inactivated, whereby one
allele is inactivated by a genetic targeting event and the other
allele is inactivated due to presence of a T-to-G point mutation at
the second base of exon 9. In a specific embodiment, a porcine
animal is provided, in which both alleles of the alpha-1,3-GT gene
are inactivated, whereby one allele is inactivated via a targeting
construct directed to Exon 9 (see, for example, FIG. 6) and the
other allele is inactivated due to presence of a T-to-G point
mutation at the second base of exon 9 (FIG. 2). Targeting, for
example, can also be directed to exon 9, and or exons 4-8.
[0041] In a further embodiment, one allele is inactivated by a
genetic targeting event and the other allele is inactivated due to
presence of a T-to-G point mutation at the second base of exon 9 of
the alpha-1,3-GT gene. In a specific embodiment, one allele is
inactivated via a targeting construct directed to Exon 9 (see, for
example, FIG. 6) and the other allele is inactivated due to
presence of a T-to-G point mutation at the second base of exon 9 of
the alpha-1,3-GT gene. In another embodiment, a method to clone
such pigs includes: enucleating an oocyte, fusing the oocyte with a
donor nucleus from a porcine cell that lacks expression of
functional alpha1,3GT, and implanting the nuclear transfer-derived
embryo into a surrogate mother.
[0042] In another embodiment, the present invention provides a
method for producing viable pigs that lack any expression of
functional alpha-1,3-GT by breeding a male pig heterozygous for the
alpha-1,3-GT gene with a female pig heterozygous for the
alpha-1,3-GT gene. In one embodiment, the pigs are heterozygous due
to the genetic modification of one allele of the alpha-1,3-GT gene
to prevent expression of that allele. In another embodiment, the
pigs are heterozygous due to the presence of a point mutation in
one allele of the alpha-1,3-GT gene. In another embodiment, the
point mutation can be a T-to-G point mutation at the second base of
exon 9 of the alpha-1,3-GT gene. In one specific embodiment, a
method to produce a porcine animal that lacks any expression of
functional alpha-1,3-GT is provided wherein a male pig that
contains a T-to-G point mutation at the second base of exon 9 of
the alpha-1,3-GT gene is bred with a female pig that contains a
T-to-G point mutation at the second base of exon 9 of the
alpha-1,3-GT gene, or vise versa.
[0043] In another aspect of the present invention, a selection
method is provided for determining whether porcine cells express
galactose alpha1,3-galactose on the cell surface. In one
embodiment, the selection procedure can be based on a bacterial
toxin to select for cells that lack expression of galactose
alpha1,3-galactose. In another embodiment, the bacterial toxin,
toxin A produced by Clostridium difficile, can be used to select
for such cells. Exposure to C. difficile toxin can cause rounding
of cells that exhibit this epitope on their surface, releasing the
cells from the plate matrix. Both targeted gene knockouts and
mutations that disable enzyme function or expression can be
detected using this selection method. Cells lacking cell surface
expression of the galactose alpha 1,3-galactose, identified using
Toxin A mediated selection described, or produced using standard
methods of gene inactivation including gene targeting, can then be
used to produce pigs that lack expression of functional
alpha1,3GT.
[0044] Other embodiments of the present invention will be apparent
to one of ordinary skill in light of the following description of
the invention, the claims and what is known in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0045] FIG. 1 is a graph depicting the relative lytic effects of
complement on cells from fetuses 680B1-4.
[0046] FIG. 2 depicts a short segment of the coding region of the
alpha-1,3-GT gene (see GenBank Acc. No. L36152) in which the point
mutation selected by Toxin A occurs. Upper sequence occurs in wild
type; lower sequence shows the change due to the point mutation in
the second allele.
[0047] FIG. 3 is a representation of a 3-dimensional model of the
UDP binding site of bovine alpha1,3GT. The aromatic ring of the
tyrosine residue (foreground, white) can be seen in close proximity
to the uracil base of UDP (grayscale).
[0048] FIG. 4 is a photograph of homozygous, alpha-1,3-GT deficient
cloned pigs produced by the methods of the invention, born on Jul.
25, 2002.
[0049] FIG. 5 is a graph depicting Anti-alpha-1,3-gal IgM levels
before and after injections of piglet islet-like cell clusters
(ICC) in alpha-1,3-GT KO mice. Each mouse received three serial ICC
injections via i.p. (200-500 ICC per injection) over 4 days. All
three recipients of wild-type (WT) piglet ICCs showed a significant
elevation of anti-alpha 1,3Gal IgM titer and subsequent return to
baseline 4 weeks after ICC implants. Sera from all three mice
injected with alpha-1,3-GT DKO piglet ICCs maintained low baseline
values of anti-alpha-1,3-gal IgM titer during the observation time
of 35 days (Phelps et al., Science 299: 411-414, 2003, figure
S4).
[0050] FIG. 6 is a diagram of the porcine alpha-1,3-GT locus,
corresponding to alpha-1,3-GT genomic sequences that can be used as
5' and 3' arms in alpha-1,3-GT knockout vectors, and the structure
of the targeted locus after homologous recombination. The names of
names and positions of the primers used for 3'PCR and long-range
PCR are indicated by short arrows. The short bar indicates the
probe used for alpha-1,3-GT Southern blot analysis. The predicted
size of Southern bands with BstEII digestion for both the
endogenous alpha-1,3-GT locus and the alpha-1,3-GT targeted locus
is also indicated.
DETAILED DESCRIPTION OF THE INVENTION
[0051] We have now proven that a viable porcine animal that lacks
any expression of functional alpha 1,3 galactosyltransferase can be
produced. The present invention provides the complete inactivation
of both alleles of the alpha 1,3 galactosyltransferase gene in
pigs, thus overcoming this longstanding hurdle and making
xenotransplantation a reality. Eliminating the expression of this
gene, resulting in a lack of galactose alpha 1,3-galactose on the
cell surface, represents the first and major step in eliminating
hyperacute rejection in pig-to-human xenotransplantation therapy.
The invention also provides organs, tissues, and cells derived from
such porcine, which are useful for xenotransplantation.
[0052] In one aspect, the invention provides porcine organs,
tissues and/or purified or substantially pure cells or cell lines
obtained from pigs that lack any expression of functional
alpha1,3GT. In another embodiment, the invention provides organs or
tissues that are useful for xenotransplantation. In a further
embodiment, the invention provides cells or cell lines that are
useful for xenotransplantation.
DEFINITIONS
[0053] As used herein, the term "animal" (as in "genetically
modified (or altered) animal") is meant to include any non-human
animal, particularly any non-human mammal, including but not
limited to pigs, sheep, goats, cattle (bovine), deer, mules,
horses, monkeys, dogs, cats, rats, mice, birds, chickens, reptiles,
fish, and insects. In one embodiment of the invention, genetically
altered pigs and methods of production thereof are provided.
[0054] As used herein, an "organ" is an organized structure, which
can be made up of one or more tissues. An "organ" performs one or
more specific biological functions. Organs include, without
limitation, heart, liver, kidney, pancreas, lung, thyroid, and
skin.
[0055] As used herein, a "tissue" is an organized structure
comprising cells and the intracellular substances surrounding them.
The "tissue", alone or in conjunction with other cells or tissues
can perform one or more biological functions.
[0056] As used herein, the terms "porcine", "porcine animal", "pig"
and "swine" are generic terms referring to the same type of animal
without regard to gender, size, or breed.
[0057] I. Genetic Targeting of the Alpha-1,3-GT Gene
[0058] In one aspect of the present invention, porcine animals are
provided in which one allele of the alpha-1,3-GT gene is
inactivated via a genetic targeting event. In another aspect of the
present invention, porcine animals are provided in which both
alleles of the alpha-1,3-GT gene are inactivated via a genetic
targeting event. In one embodiment, the gene can be targeted via
homologous recombination. In other embodiments, the gene can be
disrupted, i.e. a portion of the genetic code can be altered,
thereby affecting transcription and/or translation of that segment
of the gene. For example, disruption of a gene can occur through
substitution, deletion ("knockout") or insertion ("knockin")
techniques. Additional genes for a desired protein or regulatory
sequence that modulate transcription of an existing sequence can be
inserted. In embodiments of the present invention, the alleles of
the alpha-1,3-GT gene are rendered inactive, such that the
resultant alpha-1,3-GT enzyme can no longer generate galactose
alpha1,3-galactose on the cell surface. In one embodiment, the
alpha-1,3-GT gene can be transcribed into RNA, but not translated
into protein. In another embodiment, the alpha-1,3-GT gene can be
transcribed in a trancated form. Such a truncated RNA can either
not be translated or can be translated into a nonfunctional
protein. In an alternative embodiment, the alpha-1,3-GT gene can be
inactivated in such a way that no transcription of the gene occurs.
In a further embodiment, the alpha-1,3-GT gene can be transcribed
and then translated into a nonfunctional protein.
[0059] Pigs that possess two inactive alleles of the alpha-1,3-GT
gene are not naturally occurring. It was surprisingly discovered
that while attempting to knockout the second allele of the
alpha-1,3-GT gene through a genetic targeting event, a point
mutation was identified, which prevented the second allele from
producing functional alpha1,3GT.
[0060] Thus, in another aspect of the present invention, the
alpha-1,3-GT gene can be rendered inactive through at least one
point mutation. In one embodiment, one allele of the alpha-1,3-GT
gene can be rendered inactive through at least one point mutation.
In another embodiment, both alleles of the alpha-1,3-GT gene can be
rendered inactive through at least one point mutation. In one
embodiment, this point mutation can occur via a genetic targeting
event. In another embodiment, this point mutation can be naturally
occurring. In a further embodiment, mutations can be induced in the
alpha-1,3-GT gene via a mutagenic agent.
[0061] In one specific embodiment the point mutation can be a
T-to-G mutation at the second base of exon 9 of the alpha-1,3-GT
gene (FIG. 2). Pigs carrying a naturally occurring point mutation
in the alpha-1,3-GT gene allow for the production of
alpha1,3GT-deficient pigs free of antibiotic-resistance genes and
thus have the potential to make a safer product for human use. In
other embodiments, at least two, at least three, at least four, at
least five, at least ten or at least twenty point mutations can
exist to render the alpha-1,3-GT gene inactive. In other
embodiments, pigs are provided in which both alleles of the
alpha-1,3-GT gene contain point mutations that prevent any
expression of functional alpha1,3GT. In a specific embodiment, pigs
are provided that contain the T-to-G mutation at the second base of
exon 9 in both alleles of the alpha-1,3-GT gene (FIG. 2).
[0062] Another aspect of the present invention provides a porcine
animal, in which both alleles of the alpha-1,3-GT gene are
inactivated, whereby one allele is inactivated by a genetic
targeting event and the other allele is inactivated via a mutation.
In one embodiment, a porcine animal is provided, in which both
alleles of the alpha-1,3-GT gene are inactivated, whereby one
allele is inactivated by a genetic targeting event and the other
allele is inactivated due to presence of a T-to-G point mutation at
the second base of exon 9. In a specific embodiment, a porcine
animal is provided, in which both alleles of the alpha-1,3-GT gene
are inactivated, whereby one allele is inactivated via a targeting
construct directed to Exon 9 (see, for example, FIG. 6) and the
other allele is inactivated due to presence of a T-to-G point
mutation at the second base of exon 9.
[0063] Types of Porcine Cells
[0064] Porcine cells that can be genetically modified can be
obtained from a variety of different organs and tissues such as,
but not limited to, skin, mesenchyme, lung, pancreas, heart,
intestine, stomach, bladder, blood vessels, kidney, urethra,
reproductive organs, and a disaggregated preparation of a whole or
part of an embryo, fetus, or adult animal. In one embodiment of the
invention, porcine cells can be selected from the group consisting
of, but not limited to, epithelial cells, fibroblast cells, neural
cells, keratinocytes, hematopoietic cells, melanocytes,
chondrocytes, lymphocytes (B and T), macrophages, monocytes,
mononuclear cells, cardiac muscle cells, other muscle cells,
granulosa cells, cumulus cells, epidermal cells, endothelial cells,
Islets of Langerhans cells, blood cells, blood precursor cells,
bone cells, bone precursor cells, neuronal stem cells, primordial
stem cells, hepatocytes, keratinocytes, umbilical vein endothelial
cells, aortic endothelial cells, microvascular endothelial cells,
fibroblasts, liver stellate cells, aortic smooth muscle cells,
cardiac myocytes, neurons, Kupffer cells, smooth muscle cells,
Schwann cells, and epithelial cells, erythrocytes, platelets,
neutrophils, lymphocytes, monocytes, eosinophils, basophils,
adipocytes, chondrocytes, pancreatic islet cells, thyroid cells,
parathyroid cells, parotid cells, tumor cells, glial cells,
astrocytes, red blood cells, white blood cells, macrophages,
epithelial cells, somatic cells, pituitary cells, adrenal cells,
hair cells, bladder cells, kidney cells, retinal cells, rod cells,
cone cells, heart cells, pacemaker cells, spleen cells, antigen
presenting cells, memory cells, T cells, B cells, plasma cells,
muscle cells, ovarian cells, uterine cells, prostate cells, vaginal
epithelial cells, sperm cells, testicular cells, germ cells, egg
cells, leydig cells, peritubular cells, sertoli cells, lutein
cells, cervical cells, endometrial cells, mammary cells, follicle
cells, mucous cells, ciliated cells, nonkeratinized epithelial
cells, keratinized epithelial cells, lung cells, goblet cells,
columnar epithelial cells, squamous epithelial cells, osteocytes,
osteoblasts, and osteoclasts.
[0065] In one alternative embodiment, embryonic stem cells can be
used. An embryonic stem cell line can be employed or embryonic stem
cells can be obtained freshly from a host, such as a porcine
animal. The cells can be grown on an appropriate fibroblast-feeder
layer or grown in the presence of leukemia inhibiting factor (LIF).
In a preferred embodiment, the porcine cells can be fibroblasts; in
one specific embodiment, the porcine cells can be fetal
fibroblasts. Fibroblast cells are a preferred somatic cell type
because they can be obtained from developing fetuses and adult
animals in large quantities. These cells can be easily propagated
in vitro with a rapid doubling time and can be clonally propagated
for use in gene targeting procedures.
[0066] Targeting Constructs
[0067] Homologous Recombination
[0068] Homologous recombination permits site-specific modifications
in endogenous genes and thus novel alterations can be engineered
into the genome. In homologous recombination, the incoming DNA
interacts with and integrates into a site in the genome that
contains a substantially homologous DNA sequence. In non-homologous
("random" or "illicit") integration, the incoming DNA is not found
at a homologous sequence in the genome but integrates elsewhere, at
one of a large number of potential locations. In general, studies
with higher eukaryotic cells have revealed that the frequency of
homologous recombination is far less than the frequency of random
integration. The ratio of these frequencies has direct implications
for "gene targeting" which depends on integration via homologous
recombination (i.e. recombination between the exogenous "targeting
DNA" and the corresponding "target DNA" in the genome).
[0069] A number of papers describe the use of homologous
recombination in mammalian cells. Illustrative of these papers are
Kucherlapati et al., Proc. Natl. Acad. Sci. USA 81:3153-3157, 1984;
Kucherlapati et al., Mol. Cell. Bio. 5:714-720, 1985; Smithies et
al, Nature 317:230-234, 1985; Wake et al., Mol. Cell. Bio.
8:2080-2089, 1985; Ayares et al., Genetics 111:375-388, 1985;
Ayares et al., Mol. Cell. Bio. 7:1656-1662, 1986; Song et al.,
Proc. Natl. Acad. Sci. USA 84:6820-6824, 1987; Thomas et al. Cell
44:419-428, 1986; Thomas and Capecchi, Cell 51: 503-512, 1987;
Nandi et al., Proc. Natl. Acad. Sci. USA 85:3845-3849, 1988; and
Mansour et al., Nature 336:348-352, 1988. Evans and Kaufman, Nature
294:146-154, 1981; Doetschman et al., Nature 330:576-578, 1987;
Thoma and Capecchi, Cell 51:503-512, 4987; Thompson et al., Cell
56:316-321, 1989.
[0070] The present invention uses homologous recombination to
inactivate the alpha-1,3-GT gene in cells, such as the porcine
cells described above. The DNA can comprise at least a portion of
the gene(s) at the particular locus with introduction of an
alteration into at least one, optionally both copies, of the native
gene(s), so as to prevent expression of functional alpha1,3GT. The
alteration can be an insertion, deletion, replacement or
combination thereof. When the alteration is introduce into only one
copy of the gene being inactivated, the cells having a single
unmutated copy of the target gene are amplified and can be
subjected to a second targeting step, where the alteration can be
the same or different from the first alteration, usually different,
and where a deletion, or replacement is involved, can be
overlapping at least a portion of the alteration originally
introduced. In this second targeting step, a targeting vector with
the same arms of homology, but containing a different mammalian
selectable markers can be used. The resulting transformants are
screened for the absence of a functional target antigen and the DNA
of the cell can be further screened to ensure the absence of a
wild-type target gene. Alternatively, homozygosity as to a
phenotype can be achieved by breeding hosts heterozygous for the
mutation.
[0071] Targeting Vectors
[0072] Modification of a targeted locus of a cell can be produced
by introducing DNA into the cells, where the DNA has homology to
the target locus and includes a marker gene, allowing for selection
of cells comprising the integrated construct. The homologous DNA in
the target vector will recombine with the chromosomal DNA at the
target locus. The marker gene can be flanked on both sides by
homologous DNA sequences, a 3' recombination arm and a 5'
recombination arm. Methods for the construction of targeting
vectors have been described in the art, see, for example, Dai et
al., Nature Biotechnology 20: 251-255, 2002; WO 00/51424, FIG.
6.
[0073] Various constructs can be prepared for homologous
recombination at a target locus. The construct can include at least
50 bp, 100 bp, 500 bp, 1 kbp, 2 kbp, 4 kbp, 5 kbp, 10 kbp, 15 kbp,
20 kbp, or 50 kbp of sequence homologous with the target locus. The
sequence can include any contiguous sequence of the porcine
alpha-1,3-GT gene (see, for example, GenBank Acc. No. L36152,
WO0130992 to The University of Pittsburgh of the Commonwealth
System of Higher Education; WO 01/123541 to Alexion, Inc.).
[0074] Various considerations can be involved in determining the
extent of homology of target DNA sequences, such as, for example,
the size of the target locus, availability of sequences, relative
efficiency of double cross-over events at the target locus and the
similarity of the target sequence with other sequences.
[0075] The targeting DNA can include a sequence in which DNA
substantially isogenic flanks the desired sequence modifications
with a corresponding target sequence in the genome to be modified.
The substantially isogenic sequence can be at least about 95%,
97-98%, 99.0-99.5%, 99.6-99.9%, or 100% identical to the
corresponding target sequence (except for the desired sequence
modifications). The targeting DNA and the target DNA preferably can
share stretches of DNA at least about 75, 150 or 500 base pairs
that are 100% identical. Accordingly, targeting DNA can be derived
from cells closely related to the cell line being targeted; or the
targeting DNA can be derived from cells of the same cell line or
animal as the cells being targeted.
[0076] The DNA constructs can be designed to modify the endogenous,
target alpha1,3GT. The homologous sequence for targeting the
construct can have one or more deletions, insertions, substitutions
or combinations thereof. The alteration can be the insertion of a
selectable marker gene fused in reading frame with the upstream
sequence of the target gene.
[0077] Suitable selectable marker genes include, but are not
limited to: genes conferring the ability to grow on certain media
substrates, such as the tk gene (thymidine kinase) or the hprt gene
(hypoxanthine phosphoribosyltransferase) which confer the ability
to grow on HAT medium (hypoxanthine, aminopterin and thymidine);
the bacterial gpt gene (guanine/xanthine phosphoribosyltransferase)
which allows growth on MAX medium (mycophenolic acid, adenine, and
xanthine) See, for example, Song, K-Y., et al. Proc. Nat'l Acad.
Sci. U.S.A. 84:6820-6824 (1987); Sambrook, J., et al., Molecular
Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1989), Chapter 16. Other examples of
selectable markers include: genes conferring resistance to
compounds such as antibiotics, genes conferring the ability to grow
on selected substrates, genes encoding proteins that produce
detectable signals such as luminescence, such as green fluorescent
protein, enhanced green fluorescent protein (eGFP). A wide variety
of such markers are known and available, including, for example,
antibiotic resistance genes such as the neomycin resistance gene
(neo) (Southern, P., and P. Berg, J. Mol. Appl. Genet. 1:327-341
(1982)); and the hygromycin resistance gene (hyg) (Nucleic Acids
Research 11:6895-6911 (1983), and Te Riele, H., et al., Nature
348:649-651 (1990)). Other selectable marker genes include:
acetohydroxyacid synthase (AHAS), alkaline phosphatase (AP), beta
galactosidase (LacZ), beta glucoronidase (GUS), chloramphenicol
acetyltransferase (CAT), green fluorescent protein (GFP), red
fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan
fluorescent protein (CFP), horseradish peroxidase (HRP), luciferase
(Luc), nopaline synthase (NOS), octopine synthase (OCS), and
derivatives thereof. Multiple selectable markers are available that
confer resistance to ampicillin, bleomycin, chloramphenicol,
gentamycin, hygromycin, kanamycin, lincomycin, methotrexate,
phosphinothricin, puromycin, and tetracycline.
[0078] Methods for the incorporation of antibiotic resistance genes
and negative selection factors will be familiar to those of
ordinary skill in the art (see, e.g., WO 99/15650; U.S. Pat. No.
6,080,576; U.S. Pat. No. 6,136,566; Niwa et al., J. Biochem.
113:343-349 (1993); and Yoshida et al., Transgenic Research
4:277-287 (1995)).
TABLE-US-00002 TABLE 1 Selectable marker genes that emit detectable
signals Patent No. Title 6,319,669 Modified green fluorescent
proteins 6,316,181 Establishment of cell lines with persistent
expression of a green fluorescent protein (GFP) using a pIRES/EGFP
DNA vector construct 6,303,373 Method of measuring plasma membrane
targeting of GLUT4 6,291,177 Assay for agents which alter G-protein
coupled receptor activity 6,284,519 Cell systems having specific
interaction of peptide binding pairs 6,284,496 DNA vector for
determining the presence of out-of-reading-frame mutations
6,280,934 Assay for agents which alter G-protein coupled receptor
activity 6,274,354 Methods using cre-lox for production of
recombinant adeno-associated viruses 6,270,958 Detection of
negative-strand RNA viruses 6,268,201 IniB, iniA and iniC genes of
mycobacteria and methods of use 6,265,548 Mutant Aequorea victoria
fluorescent proteins having increased cellular fluorescence
6,261,760 Regulation of the cell cycle by sterols 6,255,558 Gene
expression 6,255,071 Mammalian viral vectors and their uses
6,251,677 Hybrid adenovirus-AAV virus and methods of use thereof
6,251,602 Cell systems having specific interaction of peptide
binding pairs 6,251,582 Alternative G-coupled receptors associated
with retroviral entry into cells, methods of identifying the same
and diagnostic and therapeutic uses thereof 6,251,384 Metastasis
models using green fluorescent protein (GFP) as a marker 6,248,558
Sequence and method for genetic engineering of proteins with cell
membrane translocating activity 6,248,550 Assays for protein
kinases using fluorescent protein substrates 6,248,543 Compositions
and methods for screening antimicrobials 6,232,107 Luciferases,
fluorescent proteins, nucleic acids encoding the luciferases and
fluorescent proteins and the use thereof in diagnostics, high
throughput screening and novelty items 6,228,639 Vectors and
methods for the mutagenesis of mammalian genes 6,225,082 Myelin
basic protein MRNA transport and translation enhancer sequences
6,221,612 Photon reducing agents for use in fluorescence assays
6,218,185 Piggybac transposon-based genetic transformation system
for insects 6,214,567 Immortalized human keratinocyte cell line
6,214,563 Photon reducing agents for reducing undesired light
emission in assays 6,210,922 Serum free production of recombinant
proteins and adenoviral vectors 6,210,910 Optical fiber biosensor
array comprising cell populations confined to microcavities
6,203,986 Visualization of RNA in living cells 6,197,928
Fluorescent protein sensors for detection of analytes 6,180,343
Green fluorescent protein fusions with random peptides 6,172,188
Fluorescent proteins 6,153,409 Process for continuous optimized
protein production in insect larvae 6,150,176 Fluorescent protein
sensors for measuring the pH of a biological sample 6,146,826 Green
fluorescent protein 6,140,132 Fluorescent protein sensors for
measuring the pH of a biological sample 6,136,539 Compositions and
methods for the inhibition of MUC-5 mucin gene expression 6,136,538
Silent inducible virus replicons and uses thereof 6,133,429
Chromophores useful for the preparation of novel tandem conjugates
6,130,313 Rapidly degrading GFP-fusion proteins 6,124,128 Long
wavelength engineered fluorescent proteins 6,110,711 Method of
defining cell types by probing comprehensive expression libraries
with amplified RNA 6,096,865 Mutants of the green fluorescent
protein having improved fluorescent properties at 37 degrees
6,096,717 Method for producing tagged genes transcripts and
proteins 6,093,808 I.kappa.B eGFP constructs, cell lines and
methods of use 6,090,919 FACS-optimized mutants of the green
fluorescent protein (GFP) 6,083,690 Methods and compositions for
identifying osteogenic agents 6,077,707 Long wavelength engineered
fluorescent proteins 6,066,476 Modified green fluorescent proteins
6,060,247 Post-mitotic neurons containing adenovirus vectors that
modulate apoptosis and growth 6,054,321 Long wavelength engineered
fluorescent proteins 6,037,133 I.kappa.B eGFP constructs, cell
lines and methods of use 6,027,881 Mutant Aequorea victoria
fluorescent proteins having increased cellular fluorescence
6,025,192 Modified retroviral vectors 6,020,192 Humanized green
fluorescent protein genes and methods 6,013,447 Random
intracellular method for obtaining optimally active nucleic acid
molecules 6,001,557 Adenovirus and methods of use thereof 5,994,077
Fluorescence-based isolation of differentially induced genes
5,994,071 Assessment of prostate cancer 5,993,778 Functional
expression of, and assay for, functional cellular receptors in vivo
5,989,808 Identification of compounds affecting specific
interaction of peptide binding pairs 5,985,577 Protein conjugates
containing multimers of green fluorescent protein 5,968,773 System
and method for regulation of gene expression 5,968,738 Two-reporter
FACS analysis of mammalian cells using green fluorescent proteins
5,958,713 Method of detecting biologically active substances by
using green fluorescent protein 5,952,236 Enzyme-based fluorescence
biosensor for chemical analysis 5,948,889 Compositions and methods
for screening antimicrobials 5,948,681 Non-viral vehicles for use
in gene transfer 5,942,387 Combinatorial process for preparing
substituted thiophene libraries 5,932,435 Screening antisense and
ribozyme nucleic acids in schizosaccharomyces pombe 5,922,576
Simplified system for generating recombinant adenoviruses 5,919,445
Use of green fluorescent protein to trace the infection of
baculovirus in insects and to increase viral UV stability 5,914,233
Screening assay for the identification of agents which alter
expression of PTH-rP
[0079] Combinations of selectable markers can also be used. For
example, to target alpha1,3GT, a neo gene (with or without its own
promoter, as discussed above) can be cloned into a DNA sequence
which is homologous to the alpha-1,3-GT gene. To use a combination
of markers, the HSV-tk gene can be cloned such that it is outside
of the targeting DNA (another selectable marker could be placed on
the opposite flank, if desired). After introducing the DNA
construct into the cells to be targeted, the cells can be selected
on the appropriate antibiotics. In this particular example, those
cells which are resistant to G418 and gancyclovir are most likely
to have arisen by homologous recombination in which the neo gene
has been recombined into the alpha-1,3-GT gene but the tk gene has
been lost because it was located outside the region of the double
crossover.
[0080] Deletions can be at least about 50 bp, more usually at least
about 100 bp, and generally not more than about 20 kbp, where the
deletion can normally include at least a portion of the coding
region including a portion of or one or more exons, a portion of or
one or more introns, and can or can not include a portion of the
flanking non-coding regions, particularly the 5'-non-coding region
(transcriptional regulatory region). Thus, the homologous region
can extend beyond the coding region into the 5'-non-coding region
or alternatively into the 3'-non-coding region. Insertions can
generally not exceed 10 kbp, usually not exceed 5 kbp, generally
being at least 50 bp, more usually at least 200 bp.
[0081] The region(s) of homology can include mutations, where
mutations can further inactivate the target gene, in providing for
a frame shift, or changing a key amino acid, or the mutation can
correct a dysfunctional allele, etc. The mutation can be a subtle
change, not exceeding about 5% of the homologous flanking
sequences. Where mutation of a gene is desired, the marker gene can
be inserted into an intron or an exon.
[0082] The construct can be prepared in accordance with methods
known in the art, various fragments can be brought together,
introduced into appropriate vectors, cloned, analyzed and then
manipulated further until the desired construct has been achieved.
Various modifications can be made to the sequence, to allow for
restriction analysis, excision, identification of probes, etc.
Silent mutations can be introduced, as desired. At various stages,
restriction analysis, sequencing, amplification with the polymerase
chain reaction, primer repair, in vitro mutagenesis, etc. can be
employed.
[0083] The construct can be prepared using a bacterial vector,
including a prokaryotic replication system, e.g. an origin
recognizable by E. coli, at each stage the construct can be cloned
and analyzed. A marker, the same as or different from the marker to
be used for insertion, can be employed, which can be removed prior
to introduction into the target cell. Once the vector containing
the construct has been completed, it can be further manipulated,
such as by deletion of the bacterial sequences, linearization,
introducing a short deletion in the homologous sequence. After
final manipulation, the construct can be introduced into the
cell.
[0084] The present invention further includes recombinant
constructs containing sequences of the alpha-1,3-GT gene. The
constructs comprise a vector, such as a plasmid or viral vector,
into which a sequence of the invention has been inserted, in a
forward or reverse orientation. The construct can also include
regulatory sequences, including, for example, a promoter, operably
linked to the sequence. Large numbers of suitable vectors and
promoters are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example. Bacterial: pBs, pQE-9 (Qiagen), phagescript, PsiX174,
pBluescript SK, pBsKS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene);
pTrc99A, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic:
pWLneo, pSv2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPv, pMSG,
pSVL (Pharmiacia), viral origin vectors (M13 vectors, bacterial
phage 1 vectors, adenovirus vectors, and retrovirus vectors), high,
low and adjustable copy number vectors, vectors which have
compatible replicons for use in combination in a single host
(pACYC184 and pBR322) and eukaryotic episomal replication vectors
(pCDM8). Other vectors include prokaryotic expression vectors such
as pcDNA II, pSL301, pSE280, pSE380, pSE420, pTrcHisA, B, and C,
pRSET A, B, and C (Invitrogen, Corp.), pGEMEX-1, and pGEMEX-2
(Promega, Inc.), the pET vectors (Novagen, Inc.), pTrc99A,
pKK223-3, the pGEX vectors, pEZZ18, pRIT2T, and pMC1871 (Pharmacia,
Inc.), pKK233-2 and pKK388-1 (Clontech, Inc.), and pProEx-HT
(Invitrogen, Corp.) and variants and derivatives thereof. Other
vectors include eukaryotic expression vectors such as pFastBac,
pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen),
pEUK-C1, pPUR, pMAM, pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, and
pYACneo (Clontech), pSVK3, pSVL, pMSG, pCH110, and pKK232-8
(Pharmacia, Inc.), p3'SS, pXT1, pSG5, pPbac, pMbac, pMC1neo, and
pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBacHis A, B, and
C, pVL1392, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3 pREP4,
pCEP4, and pEBVHis (Invitrogen, Corp.) and variants or derivatives
thereof. Additional vectors that can be used include: pUC18, pUC19,
pBlueScript, pSPORT, cosmids, phagemids, YAC's (yeast artificial
chromosomes), BAC's (bacterial artificial chromosomes), P1
(Escherichia coli phage), pQE70, pQE60, pQE9 (quagan), pBS vectors,
PhageScript vectors, BlueScript vectors, pNH8A, pNH16A, pNH18A,
pNH46A (Stratagene), pcDNA3 (Invitrogen), pGEX, pTrsfus, pTrc99A,
pET-5, pET-9, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia),
pSPORT1, pSPORT2, pCMVSPORT2.0 and pSV-SPORT1 (Invitrogen),
pTrxFus, pThioHis, pLEX, pTrcHis, pTrcHis2, pRSET, pBlueBacHis2,
pcDNA3.1/His, pcDNA3.1(-)/Myc-His, pSecTag, pEBVHis, pPIC9K,
pPIC3.5K, pA0815, pPICZ, pPICZ.quadrature., pGAPZ,
pGAPZ.quadrature., pBlueBac4.5, pBlueBacHis2, pMelBac, pSinRep5,
pSinHis, pIND, pIND(SP1), pVgRXR, pcDNA2.1, pYES2, pZErO1.1,
pZErO-2.1, pCR-Blunt, pSE280, pSE380, pSE420, pVL1392, pVL1393,
pCDM8, pcDNA1.1, pcDNA1.1/Amp, pcDNA3.1, pcDNA3.1/Zeo, pSe, SV2,
pRc/CMV2, pRc/RSV, pREP4, pREP7, pREP8, pREP9, pREP 10, pCEP4,
pEBVHis, pCR3.1, pCR2.1, pCR3.1-Uni, and pCRBac from Invitrogen;
.quadrature. ExCell, .quadrature. gt11, pTrc99A, pKK223-3,
pGEX-1.quadrature.T, pGEX-2T, pGEX-2TK, pGEX-4T-1, pGEX-4T-2,
pGEX-4T-3, pGEX-3X, pGEX-5X-1, pGEX-5X-2, pGEX-5X-3, pEZZ18,
pRIT2T, pMC1871, pSVK3, pSVL, pMSG, pCH110, pKK232-8, pSL1180,
pNEO, and pUC4K from Pharmacia; pSCREEN-1b(+), pT7Blue(R),
pT7Blue-2, pCITE-4-abc(+), pOCUS-2, pTAg, pET-32LIC, pET-30LIC,
pBAC-2cp LIC, pBACgus-2cp LIC, pT7Blue-2 LIC, pT7Blue-2,
.quadrature.SCREEN-1, .quadrature.BlueSTAR, pET-3abcd, pET-7abc,
pET9abcd, pET11abcd, pET12abc, pET-14b, pET-15b, pET-16b,
pET-17b-pET-17xb, pET-19b, pET-20b(+), pET-21abcd(+), pET-22b(+),
pET-23abcd(+), pET-24abcd(+), pET-25b(+), pET-26b(+), pET-27b(+),
pET-28abc(+), pET-29abc(+), pET-30abc(+), pET-31b(+), pET-32abc(+),
pET-33b(+), pBAC-1, pBACgus-1, pBAC4x-1, pBACgus4x-1, pBAC-3cp,
pBACgus-2cp, pBACsurf-1, plg, Signal plg, pYX, Selecta Vecta-Neo,
Selecta Vecta-Hyg, and Selecta Vecta-Gpt from Novagen; pLexA,
pB42AD, pGBT9, pAS2-1, pGAD424, pACT2, pGAD GL, pGAD GH, pGAD10,
pGilda, pEZM3, pEGFP, pEGFP-1, pEGFP-N, pEGFP-C, pEBFP, pGFPuv,
pGFP, p6xHis-GFP, pSEAP2-Basic, pSEAP2-Contral, pSEAP2-Promoter,
pSEAP2-Enhancer, p.quadrature.gal-Basic, p.quadrature.gal-Control,
p.quadrature.gal-Promoter, p.quadrature.gal-Enhancer,
pCMV.quadrature., pTet-Off, pTet-On, pTK-Hyg, pRetro-Off,
pRetro-On, pIRES1 neo, pIRES1hyg, pLXSN, pLNCX, pLAPSN, pMAMneo,
pMAMneo-CAT, pMAMneo-LUC, pPUR, pSV2neo, pYEX4T-1/2/3, pYEX-S1,
pBacPAK-His, pBacPAK8/9, pAcUW31, BacPAK6, pTriplEx,
.quadrature.gt10, .quadrature.gt11, pWE15, and .quadrature.TriplEx
from Clontech; Lambda ZAP II, pBK-CMV, pBK-RSV, pBluescript II KS
+/-, pBluescript II SK +/-, pAD-GAL4, pBD-GAL4 Cam, pSurfscript,
Lambda FIX II, Lambda DASH, Lambda EMBL3, Lambda EMBL4, SuperCos,
pCR-Scrigt Amp, pCR-Script Cam, pCR-Script Direct, pBS +/-, pBC KS
+/-, pBC SK +/-, Phagescript, pCAL-n-EK, pCAL-n, pCAL-c, pCAL-kc,
pET-3abcd, pET-11abcd, pSPUTK, pESP-1, pCMVLacI, pOPRSVUMCS, pOPI3
CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pMC1neo Poly A, pOG44,
pOG45, pFRT.quadrature.GAL, pNEO.quadrature.GAL, pRS403, pRS404,
pRS405, pRS406, pRS413, pRS414, pRS415, and pRS416 from Stratagene
and variants or derivatives thereof. Two-hybrid and reverse
two-hybrid vectors can also be used, for example, pPC86, pDBLeu,
pDBTrp, pPC97, p2.5, pGAD1-3, pGAD10, pACt, pACT2, pGADGL, pGADGH,
pAS2-1, pGAD424, pGBT8, pGBT9, pGAD-GAL4, pLexA, pBD-GAL4, pHISi,
pHISi-1, placZi, pB42AD, pDG202, pJK202, pJG4-5, pNLexA, pYESTrp
and variants or derivatives thereof. Any other plasmids and vectors
may be used as long as they are replicable and viable in the
host.
[0085] Techniques which can be used to allow the DNA construct
entry into the host cell include calcium phosphate/DNA co
precipitation, microinjection of DNA into the nucleus,
electroporation, bacterial protoplast fusion with intact cells,
transfection, or any other technique known by one skilled in the
art. The DNA can be single or double stranded, linear or circular,
relaxed or supercoiled DNA. For various techniques for transfecting
mammalian cells, see, for example, Keown et al., Methods in
Enzymology Vol. 185, pp. 527-537 (1990).
[0086] In one specific embodiment, heterozygous knockout cells can
be produced by transfection of primary porcine fetal fibroblasts
with a knockout vector containing alpha-1,3-GT sequence isolated
from isogenic DNA. As described in Dai et al. (Nature
Biotechnology, 20:451-455), the 5' arm can be 4.9 kb and be
comprised of a large fragment of intron 8 and the 5' end of exon 9.
The 3' arm can be and be comprised of exon 9 sequence. The vector
can incorporate a promoter trap strategy, using, for example, IRES
(internal ribosome entry site) to initiate translation of the Neor
gene (see, for example, FIG. 6).
[0087] Selection of Homologously Recombined Cells
[0088] The cells can then be grown in appropriately-selected medium
to identify cells providing the appropriate integration. The
presence of the selectable marker gene inserted into the
alpha-1,3-GT gene establishes the integration of the target
construct into the host genome. Those cells which show the desired
phenotype can then be further analyzed by restriction analysis,
electrophoresis, Southern analysis, polymerase chain reaction, etc
to analyze the DNA in order to establish whether homologous or
non-homologous recombination occurred. This can be determined by
employing probes for the insert and then sequencing the 5' and 3'
regions flanking the insert for the presence of the alpha-1,3-GT
gene extending beyond the flanking regions of the construct or
identifying the presence of a deletion, when such deletion is
introduced. Primers can also be used which are complementary to a
sequence within the construct and complementary to a sequence
outside the construct and at the target locus. In this way, one can
only obtain DNA duplexes having both of the primers present in the
complementary chains if homologous recombination has occurred. By
demonstrating the presence of the primer sequences or the expected
size sequence, the occurrence of homologous recombination is
supported.
[0089] The polymerase chain reaction used for screening homologous
recombination events is known in the art, see, for example, Kim and
Smithies, Nucleic Acids Res. 16:8887-8903, 1988; and Joyner et al.,
Nature 338:153-156, 1989. The specific combination of a mutant
polyoma enhancer and a thymidine kinase promoter to drive the
neomycin gene has been shown to be active in both embryonic stem
cells and EC cells by Thomas and Capecchi, supra, 1987; Nicholas
and Berg (1983) in Teratocarcinoma Stem Cell, eds. Siver, Martin
and Strikland (Cold Spring Harbor Lab., Cold Spring Harbor, N.Y.
(pp. 469-497); and Linney and Donerly, Cell 35:693-699, 1983.
[0090] The cell lines obtained from the first round of targeting
are likely to be heterozygous for the targeted allele.
Homozygosity, in which both alleles are modified, can be achieved
in a number of ways. One approach is to grow up a number of cells
in which one copy has been modified and then to subject these cells
to another round of targeting using a different selectable marker.
Alternatively, homozygotes can be obtained by breeding animals
heterozygous for the modified allele, according to traditional
Mendelian genetics. In some situations, it can be desirable to have
two different modified alleles. This can be achieved by successive
rounds of gene targeting or by breeding heterozygotes, each of
which carries one of the desired modified alleles.
[0091] Induced Mutation in the Alpha 1,3 GT Locus
[0092] In certain other embodiments, the methods of the invention
involve the intentional introduction of a mutation via a mutagenic
agent. Examples of mutagenic agents known in the art and suitable
for use in the present invention include, but are not limited to,
chemical mutagens (e.g., DNA-intercalating or DNA-binding chemicals
such as N-ethyl-N-nitrosourea (ENU), ethylmethanesulphonate (EMS),
mustard gas, ICR191 and the like; see, e.g., E. C. Friedberg, G. C.
Walker, W. Siede, DNA Repair and Mutagenesis, ASM Press, Washington
D.C. (1995), physical mutagens (e.g., UV radiation, radiation,
x-rays), biochemical mutagens (e.g., restriction enzymes, DNA
repair mutagens, DNA repair inhibitors, and error-prone DNA
polymerases and replication proteins), as well as transposon
insertion. According to the methods of the present invention, cells
in culture can be exposed to one of these agents, and any mutation
resulting in the depletion of galactose alpha1,3-galactose on the
cell surface can be selected, for example, via exposure to toxin
A.
[0093] Preferred doses of chemical mutagens for inducing mutations
in cells are known in the art, or can be readily determined by the
ordinarily skilled artisan using assays of mutagenesis known in the
art. Chemical mutagenesis of cells in vitro can be achieved by
treating the cells with various doses of the mutagenic agent and/or
controlling the time of exposure to the agent. By titrating the
mutagenic agent exposure and/or dose, it is possible to carry out
the optimal degree of mutagenesis for the intended purpose, thereby
mutating a desired number of genes in each target cell. For
example, useful doses of ENU can be 0.1-0.4 mg/ml for approximately
1-2 hours. In another example, useful doses of EMS can be 0.1-1
mg/ml for approximately 10-30 hours. In addition, lower and higher
doses and exposure times can also be used to achieve the desired
mutation frequency.
[0094] II. Identification of Cells that do not Express Functional
Alpha-1,3-GT
[0095] In another aspect of the present invention, a selection
method is provided for determining whether porcine cells lack
expression of functional alpha-1,3-GT.
[0096] In one embodiment, the selection procedure can be based on a
bacterial toxin to select for cells that lack expression of
functional alpha1,3GT. In another embodiment, the bacterial toxin,
toxin A produced by Clostridium difficile, can be used to select
for cells lacking the cell surface epitope galactose
alpha1,3-galactose. Exposure to C. difficile toxin can cause
rounding of cells that exhibit this epitope on their surface,
releasing the cells from the plate matrix. Both targeted gene
knockouts and mutations that disable enzyme function or expression
can be detected using this selection method. Cells lacking cell
surface expression of the galactose alpha 1,3-galactose epitope,
identified using Toxin A mediated selection described, or produced
using standard methods of gene inactivation including gene
targeting, can then be used to produce pigs, in which both alleles
of the alpha 1,3 GT gene are inactive.
[0097] In one embodiment, the selection method can detect the
depletion of the alpha 1,3GT epitope directly, whether due to
targeted knockout of the alpha 1,3GT gene by homologous
recombination, or a mutation in the gene that results in a
nonfunctioning or nonexpressed enzyme. Selection via antibiotic
resistance has been used most commonly for screening (see above).
This method can detect the presence of the resistance gene on the
targeting vector, but does not directly indicate whether
integration was a targeted recombination event or a random
integration. Certain technology, such as Poly A and promoter trap
technology, increase the probability of targeted events, but again,
do not give direct evidence that the desired phenotype, a cell
deficient in gal alpha 1,3 gal epitopes on the cell surface, has
been achieved. In addition, negative forms of selection can be used
to select for targeted integration; in these cases, the gene for a
factor lethal to the cells is inserted in such a way that only
targeted events allow the cell to avoid death. Cells selected by
these methods can then be assayed for gene disruption, vector
integration and, finally, alpha 1,3gal epitope depletion. In these
cases, since the selection is based on detection of targeting
vector integration and not at the altered phenotype, only targeted
knockouts, not point mutations, gene rearrangements or truncations
or other such modifications can be detected.
[0098] Toxin A, a cytotoxin produced by the bacterium Clostridium
difficile, specifically binds the terminal carbohydrate glactose
alpha1,3-galactose sequence gal alpha 1-3gal beta 1-4GlcNAc.
Binding to this receptor mediates a cytotoxic effect on the cell,
causing it to change morphology and, in some cases, to release from
the plate matrix. Under controlled conditions, cells not carrying
this marker are unaffected by the toxin. Thus, in one embodiment,
to determine whether or not the alpha 1,3 gal epitope has been
successfully eliminated via targeted knockout or gene mutation of
the gal alpha-1,3-GT locus, cells that do not carry the epitope can
be selected. Exposure to toxin A can be toxic for cells carrying
the epitope, and promote selection for those cells in which the
gene has been successfully inactivated. Thus, according to on
aspect of the present invention, cells useful as nuclear donors for
production of genetically altered animals (e.g., pigs) that are
knocked out or mutated in the gal alpha 1,3 locus are selected by
exposure of cells to C. difficile toxin A.
[0099] Toxin A, one of two cytotoxins produced by Clostridium
difficile, has a high binding affinity for the galactose
alpha1,3-galactose sequence gal alpha 1,3-gal beta 1,4GlcNAc found
on the surface of a variety of cell types (Clark et al., Arch.
Biochem. Biophys. 257 (1): 217-229, 1987). This carbohydrate seems
to serve as a functional receptor for Toxin A, as cells displaying
this epitope on their surface are more sensitive to the cytotoxic
effect of toxin A than are cells lacking this receptor. Sensitive
cells exposed to toxin A in culture exhibit cell rounding, probably
due to actin depolymerization and resultant changes in cytoskeletal
integrity (Kushnaryov et al., J. Biol. Chem. 263: 17755-17762
(1988)and Just et al., J. Clin. Invest. 95: 1026-1031, 1995). These
cells can be selectively removed from the culture, as they lift
from the matrix and float in suspension, leaving unaffected cells
firmly attached to the plate surface.
[0100] Exposure of cells to toxin A. In one embodiment, attached
cells are exposed to toxin A as a component of cell culture media.
After a fixed time of exposure, the media containing the toxin A
and released toxin A-sensitive cells are removed, the plate washed,
and the media, without toxin A, replenished. The exposure to toxin
A is repeated over a period of days to remove attached
toxin-sensitive cells from the plates, and allow insensitive cells
to proliferate and expand. Purified toxin A can be used in the
methods of the present invention (available commercially, see for
example, Techlab Inc., Cat. #T3001, Blacksburg, Va.). Crude
unpurified toxin A can also be used (available commercially, see
for example, Techlab Inc. Cat. #T5000 or T3000, Blacksburg, Va.),
which can require initial titering to determine effective dosage
for selection.
[0101] Serum-Based Selection Method
[0102] In another embodiment, the selection procedure can be
conducted using serum containing complement factors and natural
antibodies to the gal alpha1,3gal epitope (see, for example, Koike
et al., Xenotransplantation 4:147-153, 1997). Exposure to serum
from a human or non-human primate that contains anti-Gal antibodies
can cause cell lysis due to specific antibody binding and
complement activation in cells that exhibit gal alpha 1,3 gal
epitope. Therefore, cells deficient in alpha-1,3-GT will remain
alive and thus can be selected.
[0103] Further Characterization of Porcine Cells Lacking Expression
of Functional Alpha1,3GT
[0104] Porcine cells believed to lacking expression of functional
alpha-1,3-GT can be further characterized. Such characterization
can be accomplished by the following techniques, including, but not
limited to: PCR analysis, Southern blot analysis, Northern blot
analysis, specific lectin binding assays, and/or sequencing
analysis.
[0105] PCR analysis as described in the art (see, for example, Dai
et al. Nature Biotechnology 20:431-455) can be used to determine
the integration of targeting vectors. In one embodiment, amplimers
can originate in the antibiotic resistance gene and extend into a
region outside the vector sequence. Southern analysis (see, for
example, Dai et al. Nature Biotechnology 20:431-455) can also be
used to characterize gross modifications in the locus, such as the
integration of a targeting vector into the alpha 1,3GT locus.
Whereas, Northern analysis can be used to characterize the
transcript produced from each of the alleles.
[0106] Specific lectin binding, using GSL IB4 lectin from Griffonia
(Bandeiraea) simplicifolia (Vector Labs), a lectin that
specifically binds the carbohydrate moiety gal alpha 1,3 gal, and
FACS (fluorescent antibody cell sorting) analysis of binding can
determine whether or not the alpha 1,3 gal epitope is present on
the cells. This type of analysis involves the addition of
fluorescein-labeled GSL-IB4 lectin to the cells and subsequent cell
sorting.
[0107] Further, sequencing analysis of the cDNA produced from the
RNA transcript can also be used to determine the precise location
of any mutations in the alpha 1,3GT allele.
[0108] III. Production of Porcine Animals
[0109] In yet another aspect, the present invention provides a
method for producing viable pigs in which both alleles of the
alpha-1,3-GT gene have been rendered inactive. In one embodiment,
the pigs are produced by cloning using a donor nucleus from a
porcine cell in which both alleles of the alpha-1,3-GT gene have
been inactivated. In one embodiment, both alleles of the
alpha-1,3-GT gene are inactivated via a genetic targeting event. In
another embodiment, both alleles of the alpha-1,3-GT gene are
inactivated due to the presence of a point mutation. In another
embodiment, one allele is inactivated by a genetic targeting event
and the other allele is inactivated via a point mutation. In a
further embodiment, one allele is inactivated by a genetic
targeting event and the other allele is inactivated due to presence
of a T-to-G point mutation at the second base of exon 9 of the
alpha-1,3-GT gene. In a specific embodiment, one allele is
inactivated via a targeting construct directed to Exon 9 (FIG. 6)
and the other allele is inactivated due to presence of a T-to-G
point mutation at the second base of exon 9 of the alpha-1,3-GT
gene. In another embodiment, a method to clone such pigs includes:
enucleating an oocyte, fusing the oocyte with a donor nucleus from
a porcine cell in which both alleles of the alpha-1,3-GT gene have
been inactivated, and implanting the nuclear transfer-derived
embryo into a surrogate mother.
[0110] Alternatively, a method is provided for producing viable
pigs that lack any expression of functional alpha-1,3-GT by
inactivating both alleles of the alpha-1,3-GT gene in embryonic
stem cells, which can then be used to produce offspring.
[0111] Genetically altered animals that can be created by modifying
zygotes directly. For mammals, the modified zygotes can be then
introduced into the uterus of a pseudopregnant female capable of
carrying the animal to term. For example, if whole animals lacking
the alpha-1,3-GT gene are desired, then embryonic stem cells
derived from that animal can be targeted and later introduced into
blastocysts for growing the modified cells into chimeric animals.
For embryonic stem cells, either an embryonic stem cell line or
freshly obtained stem cells can be used.
[0112] In a suitable embodiment of the invention, the totipotent
cells are embryonic stem (ES) cells. The isolation of ES cells from
blastocysts, the establishing of ES cell lines and their subsequent
cultivation are carried out by conventional methods as described,
for example, by Doetchmann et al., J. Embryol. Exp. Morph. 87:27-45
(1985); Li et al., Cell 69:915-926 (1992); Robertson, E. J.
"Tetracarcinomas and Embryonic Stem Cells: A Practical Approach,"
ed. E. J. Robertson, IRL Press, Oxford, England (1987); Wurst and
Joyner, "Gene Targeting: A Practical Approach," ed. A. L. Joyner,
IRL Press, Oxford, England (1993); Hogen et al., "Manipulating the
Mouse Embryo: A Laboratory Manual," eds. Hogan, Beddington,
Costantini and Lacy, Cold Spring Harbor Laboratory Press, New York
(1994); and Wang et al., Nature 336:741-744 (1992). In another
suitable embodiment of the invention, the totipotent cells are
embryonic germ (EG) cells. Embryonic Germ cells are
undifferentiated cells functionally equivalent to ES cells, that is
they can be cultured and transfected in vitro, then contribute to
somatic and germ cell lineages of a chimera (Stewart et al., Dev.
Biol. 161:626-628 (1994)). EG cells are derived by culture of
primordial germ cells, the progenitors of the gametes, with a
combination of growth factors: leukemia inhibitory factor, steel
factor and basic fibroblast growth factor (Matsui et al., Cell
70:841-847 (1992); Resnick et al., Nature 359:550-551 (1992)). The
cultivation of EG cells can be carried out using methods described
in the article by Donovan et al., "Transgenic Animals, Generation
and Use," Ed. L. M. Houdebine, Harwood Academic Publishers (1997),
and in the original literature cited therein.
[0113] Tetraploid blastocysts for use in the invention may be
obtained by natural zygote production and development, or by known
methods by electrofusion of two-cell embryos and subsequently
cultured as described, for example, by James et al., Genet. Res.
Camb. 60:185-194 (1992); Nagy and Rossant, "Gene Targeting: A
Practical Approach," ed. A. L. Joyner, IRL Press, Oxford, England
(1993); or by Kubiak and Tarkowski, Exp. Cell Res. 157:561-566
(1985).
[0114] The introduction of the ES cells or EG cells into the
blastocysts can be carried out by any method known in the art. A
suitable method for the purposes of the present invention is the
microinjection method as described by Wang et al., EMBO J.
10:2437-2450 (1991).
[0115] Alternatively, by modified embryonic stem cells transgenic
animals can be produced. The genetically modified embryonic stem
cells can be injected into a blastocyst and then brought to term in
a female host mammal in accordance with conventional techniques.
Heterozygous progeny can then be screened for the presence of the
alteration at the site of the target locus, using techniques such
as PCR or Southern blotting. After mating with a wild-type host of
the same species, the resulting chimeric progeny can then be
cross-mated to achieve homozygous hosts.
[0116] After transforming embryonic stem cells with the targeting
vector to alter the alpha-1,3-GT gene, the cells can be plated onto
a feeder layer in an appropriate medium, e.g., fetal bovine serum
enhanced DMEM. Cells containing the construct can be detected by
employing a selective medium, and after sufficient time for
colonies to grow, colonies can be picked and analyzed for the
occurrence of homologous recombination. Polymerase chain reaction
can be used, with primers within and without the construct sequence
but at the target locus. Those colonies which show homologous
recombination can then be used for embryo manipulating and
blastocyst injection. Blastocysts can be obtained from
superovulated females. The embryonic stem cells can then be
trypsinized and the modified cells added to a droplet containing
the blastocysts. At least one of the modified embryonic stem cells
can be injected into the blastocoel of the blastocyst. After
injection, at least one of the blastocysts can be returned to each
uterine horn of pseudopregnant females. Females are then allowed to
go to term and the resulting litters screened for mutant cells
having the construct. The blastocysts are selected for different
parentage from the transformed ES cells. By providing for a
different phenotype of the blastocyst and the ES cells, chimeric
progeny can be readily detected, and then genotyping can be
conducted to probe for the presence of the modified alpha-1,3-GT
gene.
[0117] Somatic Cell Nuclear Transfer to Produce Cloned, Transgenic
Offspring
[0118] The present invention provides a method for cloning a pig
lacking a functional alpha-1,3-GT gene via somatic cell nuclear
transfer. In general, the pig can be produced by a nuclear transfer
process comprising the following steps: obtaining desired
differentiated pig cells to be used as a source of donor nuclei;
obtaining oocytes from a pig; enucleating said oocytes;
transferring the desired differentiated cell or cell nucleus into
the enucleated oocyte, e.g., by fusion or injection, to form NT
units; activating the resultant NT unit; and transferring said
cultured NT unit to a host pig such that the NT unit develops into
a fetus.
[0119] Nuclear transfer techniques or nuclear transplantation
techniques are known in the art(Dai et al. Nature Biotechnology
20:251-255; Polejaeva et al Nature 407:86-90 (2000); Campbell et
al, Theriogenology, 43:181 (1995); Collas et al, Mol. Report. Dev.,
38:264-267 (1994); Keefer et al, Biol. Reprod., 50:935-939 (1994);
Sims et al, Proc. Natl. Acad. Sci., USA, 90:6143-6147 (1993); WO
94/26884; WO 94/24274, and WO 90/03432, U.S. Pat. Nos. 4,944,384
and 5,057,420).
[0120] A donor cell nucleus, which has been modified to alter the
alpha-1,3-GT gene, is transferred to a recipient porcine oocyte.
The use of this method is not restricted to a particular donor cell
type. The donor cell can be as described herein, see also, for
example, Wilmut et al Nature 385 810 (1997); Campbell et al Nature
380 64-66 (1996); Dai et al., Nature Biotechnology 20:251-255, 2002
or Cibelli et al Science 280 1256-1258 (1998). All cells of normal
karyotype, including embryonic, fetal and adult somatic cells which
can be used successfully in nuclear transfer can be employed. Fetal
fibroblasts are a particularly useful class of donor cells.
Generally suitable methods of nuclear transfer are described in
Campbell et al Theriogenology 43 181 (1995), Dai et al. Nature
Biotechnology 20:251-255, Polejaeva et al Nature 407:86-90 (2000),
Collas et al Mol. Reprod. Dev. 38 264-267 (1994), Keefer et al
Biol. Reprod. 50 935-939 (1994), Sims et al Proc. Nat'l. Acad. Sci.
USA 90 6143-6147 (1993), WO-A-9426884, WO-A-9424274, WO-A-9807841,
WO-A-9003432, U.S. Pat. No. 4,994,384 and U.S. Pat. No. 5,057,420.
Differentiated or at least partially differentiated donor cells can
also be used. Donor cells can also be, but do not have to be, in
culture and can be quiescent. Nuclear donor cells which are
quiescent are cells which can be induced to enter quiescence or
exist in a quiescent state in vivo. Prior art methods have also
used embryonic cell types in cloning procedures (Campbell et al
(Nature, 380:64-68, 1996) and Stice et al (Biol. Reprod., 20
54:100-110, 1996).
[0121] Somatic nuclear donor cells may be obtained from a variety
of different organs and tissues such as, but not limited to, skin,
mesenchyme, lung, pancreas, heart, intestine, stomach, bladder,
blood vessels, kidney, urethra, reproductive organs, and a
disaggregated preparation of a whole or part of an embryo, fetus,
or adult animal. In a suitable embodiment of the invention, nuclear
donor cells are selected from the group consisting of epithelial
cells, fibroblast cells, neural cells, keratinocytes, hematopoietic
cells, melanocytes, chondrocytes, lymphocytes (B and T),
macrophages, monocytes, mononuclear cells, cardiac muscle cells,
other muscle cells, granulosa cells, cumulus cells, epidermal cells
or endothelial cells. In another embodiment, the nuclear donor cell
is an embryonic stem cell. In a preferred embodiment, fibroblast
cells can be used as donor cells.
[0122] In another embodiment of the invention, the nuclear donor
cells of the invention are germ cells of an animal. Any germ cell
of an animal species in the embryonic, fetal, or adult stage may be
used as a nuclear donor cell. In a suitable embodiment, the nuclear
donor cell is an embryonic germ cell.
[0123] Nuclear donor cells may be arrested in any phase of the cell
cycle (G0, G1, G2, S, M) so as to ensure coordination with the
acceptor cell. Any method known in the art may be used to
manipulate the cell cycle phase. Methods to control the cell cycle
phase include, but are not limited to, G0 quiescence induced by
contact inhibition of cultured cells, G0 quiescence induced by
removal of serum or other essential nutrient, G0 quiescence induced
by senescence, G0 quiescence induced by addition of a specific
growth factor; G0 or G1 quiescence induced by physical or chemical
means such as heat shock, hyperbaric pressure or other treatment
with a chemical, hormone, growth factor or other substance; S-phase
control via treatment with a chemical agent which interferes with
any point of the replication procedure; M-phase control via
selection using fluorescence activated cell sorting, mitotic shake
off, treatment with microtubule disrupting agents or any chemical
which disrupts progression in mitosis (see also Freshney, R. I.,
"Culture of Animal Cells: A Manual of Basic Technique," Alan R.
Liss, Inc, New York (1983).
[0124] Methods for isolation of oocytes are well known in the art.
Essentially, this can comprise isolating oocytes from the ovaries
or reproductive tract of a pig. A readily available source of pig
oocytes is slaughterhouse materials. For the combination of
techniques such as genetic engineering, nuclear transfer and
cloning, oocytes must generally be matured in vitro before these
cells can be used as recipient cells for nuclear transfer, and
before they can be fertilized by the sperm cell to develop into an
embryo. This process generally requires collecting immature
(prophase I) oocytes from mammalian ovaries, e.g., bovine ovaries
obtained at a slaughterhouse, and maturing the oocytes in a
maturation medium prior to fertilization or enucleation until the
oocyte attains the metaphase II stage, which in the case of bovine
oocytes generally occurs about 18-24 hours post-aspiration. This
period of time is known as the "maturation period". In certain
embodiments, the oocyte is obtained from a gilt. A "gilt" is a
female pig that has never had offspring. In other embodiments, the
oocyte is obtained from a sow. A "sow" is a female pig that has
previously produced offspring.
[0125] A metaphase II stage oocyte can be the recipient oocyte, at
this stage it is believed that the oocyte can be or is sufficiently
"activated" to treat the introduced nucleus as it does a
fertilizing sperm. Metaphase II stage oocytes, which have been
matured in vivo have been successfully used in nuclear transfer
techniques. Essentially, mature metaphase II oocytes can be
collected surgically from either non-superovulated or superovulated
porcine 35 to 48, or 39-41, hours past the onset of estrus or past
the injection of human chorionic gonadotropin (hCG) or similar
hormone.
[0126] After a fixed time maturation period, which ranges from
about 10 to 40 hours, and preferably about 16-18 hours, the oocytes
can be enucleated. Prior to enucleation the oocytes can be removed
and placed in appropriate medium, such as HECM containing 1
milligram per milliliter of hyaluronidase prior to removal of
cumulus cells. The stripped oocytes can then be screened for polar
bodies, and the selected metaphase II oocytes, as determined by the
presence of polar bodies, are then used for nuclear transfer.
Enucleation follows.
[0127] Enucleation can be performed by known methods, such as
described in U.S. Pat. No. 4,994,384. For example, metaphase II
oocytes can be placed in either HECM, optionally containing 7.5
micrograms per milliliter cytochalasin B, for immediate
enucleation, or can be placed in a suitable medium, for example an
embryo culture medium such as CR1aa, plus 10% estrus cow serum, and
then enucleated later, preferably not more than 24 hours later, and
more preferably 16-18 hours later.
[0128] Enucleation can be accomplished microsurgically using a
micropipette to remove the polar body and the adjacent cytoplasm.
The oocytes can then be screened to identify those of which have
been successfully enucleated. One way to screen the oocytes is to
stain the oocytes with 1 microgram per milliliter 33342 Hoechst dye
in HECM, and then view the oocytes under ultraviolet irradiation
for less than 10 seconds. The oocytes that have been successfully
enucleated can then be placed in a suitable culture medium, for
example, CR1aa plus 10% serum.
[0129] A single mammalian cell of the same species as the
enucleated oocyte can then be transferred into the perivitelline
space of the enucleated oocyte used to produce the NT unit. The
mammalian cell and the enucleated oocyte can be used to produce NT
units according to methods known in the art. For example, the cells
can be fused by electrofusion. Electrofusion is accomplished by
providing a pulse of electricity that is sufficient to cause a
transient breakdown of the plasma membrane. This breakdown of the
plasma membrane is very short because the membrane reforms rapidly.
Thus, if two adjacent membranes are induced to breakdown and upon
reformation the lipid bilayers intermingle, small channels can open
between the two cells. Due to the thermodynamic instability of such
a small opening, it enlarges until the two cells become one. See,
for example, U.S. Pat. No. 4,997,384 by Prather et al. A variety of
electrofusion media can be used including, for example, sucrose,
mannitol, sorbitol and phosphate buffered solution. Fusion can also
be accomplished using Sendai virus as a fusogenic agent (Graham,
Wister Inot. Symp. Monogr., 9, 19, 1969). Also, the nucleus can be
injected directly into the oocyte rather than using electroporation
fusion. See, for example, Collas and Barnes, Mol. Reprod. Dev.,
38:264-267 (1994). After fusion, the resultant fused NT units are
then placed in a suitable medium until activation, for example,
CR1aa medium. Typically activation can be effected shortly
thereafter, for example less than 24 hours later, or about 4-9
hours later, or optimally 1-2 hours after fusion. In a preferred
embodiments, activation occurs at least one hour post fusion and at
40-41 hours post maturation.
[0130] The NT unit can be activated by known methods. Such methods
include, for example, culturing the NT unit at sub-physiological
temperature, in essence by applying a cold, or actually cool
temperature shock to the NT unit. This can be most conveniently
done by culturing the NT unit at room temperature, which is cold
relative to the physiological temperature conditions to which
embryos are normally exposed. Alternatively, activation can be
achieved by application of known activation agents. For example,
penetration of oocytes by sperm during fertilization has been shown
to activate prefusion oocytes to yield greater numbers of viable
pregnancies and multiple genetically identical calves after nuclear
transfer. Also, treatments such as electrical and chemical shock
can be used to activate NT embryos after fusion. See, for example,
U.S. Pat. No. 5,496,720, to Susko-Parrish et al. Additionally,
activation can be effected by simultaneously or sequentially by
increasing levels of divalent cations in the oocyte, and reducing
phosphorylation of cellular proteins in the oocyte. This can
generally be effected by introducing divalent cations into the
oocyte cytoplasm, e.g., magnesium, strontium, barium or calcium,
e.g., in the form of an ionophore. Other methods of increasing
divalent cation levels include the use of electric shock, treatment
with ethanol and treatment with caged chelators. Phosphorylation
can be reduced by known methods, for example, by the addition of
kinase inhibitors, e.g., serine-threonine kinase inhibitors, such
as 6-dimethyl-aminopurine, staurosporine, 2-aminopurine, and
sphingosine. Alternatively, phosphorylation of cellular proteins
can be inhibited by introduction of a phosphatase into the oocyte,
e.g., phosphatase 2A and phosphatase 2B.
[0131] The activated NT units, or "fused embyos", can then be
cultured in a suitable in vitro culture medium until the generation
of cell colonies. Culture media suitable for culturing and
maturation of embryos are well known in the art. Examples of known
media, which can be used for embryo culture and maintenance,
include Ham's F-10+10% fetal calf serum (FCS), Tissue Culture
Medium-199 (TCM-199)+10% fetal calf serum,
Tyrodes-Albumin-Lactate-Pyruvate (TALP), Dulbecco's Phosphate
Buffered Saline (PBS), Eagle's and Whitten's media, and, in one
specific example, the activated NT units can be cultured in NCSU-23
medium for about 1-4 h at approximately 38.6.degree. C. in a
humidified atmosphere of 5% CO2.
[0132] Afterward, the cultured NT unit or units can be washed and
then placed in a suitable media contained in well plates which
preferably contain a suitable confluent feeder layer. Suitable
feeder layers include, by way of example, fibroblasts and
epithelial cells. The NT units are cultured on the feeder layer
until the NT units reach a size suitable for transferring to a
recipient female, or for obtaining cells which can be used to
produce cell colonies. Preferably, these NT units can be cultured
until at least about 2 to 400 cells, about 4 to 128 cells, or at
least about 50 cells.
[0133] Activated NT units can then be transferred (embryo
transfers) to the oviduct of an female pigs. In one embodiment, the
female pigs can be an estrus-synchronized recipient gilt. Crossbred
gilts (large white/Duroc/Landrace) (280-400 lbs) can be used. The
gilts can be synchronized as recipient animals by oral
administration of 18-20 mg Regu-Mate (Altrenogest, Hoechst, Warren,
N.J.) mixed into the feed. Regu-Mate can be fed for 14 consecutive
days. One thousand units of Human Chorionic Gonadotropin (hCG,
Intervet America, Millsboro, Del.) can then be administered i.m.
about 105 h after the last Regu-Mate treatment. Embryo transfers of
the can then be performed about 22-26 h after the hCG injection. In
one embodiment, the pregnancy can be brought to term and result in
the birth of live offspring. In another embodiment, the pregnancy
can be terminated early and embryonic cells can be harvested.
[0134] Breeding for Desired Homozygous Knockout Animals
[0135] In another aspect, the present invention provides a method
for producing viable pigs that lack any expression of functional
alpha-1,3-GT is provided by breeding a male pig heterozygous for
the alpha-1,3-GT gene with a female pig heterozygous for the
alpha-1,3-GT gene. In one embodiment, the pigs are heterozygous due
to the genetic modification of one allele of the alpha-1,3-GT gene
to prevent expression of that allele. In another embodiment, the
pigs are heterozygous due to the presence of a point mutation in
one allele of the alpha-1,3-GT gene. In another embodiment, the
point mutation can be a T-to-G point mutation at the second base of
exon 9 of the alpha-1,3-GT gene. In one specific embodiment, a
method to produce a porcine animal that lacks any expression of
functional alpha-1,3-GT is provided wherein a male pig that
contains a T-to-G point mutation at the second base of exon 9 of
the alpha-1,3-GT gene is bred with a female pig that contains a
T-to-G point mutation at the second base of exon 9 of the
alpha-1,3-GT gene.
[0136] In one embodiment, sexually mature animals produced from
nuclear transfer from donor cells that carrying a double knockout
in the alpha-1,3-GT gene, can be bred and their offspring tested
for the homozygous knockout. These homozygous knockout animals can
then be bred to produce more animals.
[0137] In another embodiment, oocytes from a sexually mature double
knockout animal can be in vitro fertilized using wild type sperm
from two genetically diverse pig lines and the embryos implanted
into suitable surrogates. Offspring from these matings can be
tested for the presence of the knockout, for example, they can be
tested by cDNA sequencing, PCR, toxin A sensitivity and/or lectin
binding. Then, at sexual maturity, animals from each of these
litters can be mated.
[0138] In certain methods according to this aspect of the
invention, pregnancies can be terminated early so that fetal
fibroblasts can be isolated and further characterized
phenotypically and/or genotypically. Fibroblasts that lack
expression of the alpha-1,3-GT gene can then be used for nuclear
transfer according to the methods described herein (see also Dai et
al.) to produce multiple pregnancies and offspring carrying the
desired double knockout.
[0139] IV. Types of Genetically Modified Porcine Animals
[0140] In one aspect of the present invention, porcine animals are
provided in which one allele of the alpha-1,3-GT gene is
inactivated via a genetic targeting event. In another aspect of the
present invention, porcine animals are provided in which both
alleles of the alpha-1,3-GT gene are inactivated via a genetic
targeting event. In one embodiment, the gene can be targeted via
homologous recombination. In other embodiments, the gene can be
disrupted, i.e. a portion of the genetic code can be altered,
thereby affecting transcription and/or translation of that segment
of the gene. For example, disruption of a gene can occur through
substitution, deletion ("knockout") or insertion ("knockin")
techniques. Additional genes for a desired protein or regulatory
sequence that modulate transcription of an existing sequence can be
inserted.
[0141] Pigs that possess two inactive alleles of the alpha-1,3-GT
gene are not naturally occurring. It was surprisingly discovered
that while attempting to knockout the second allele of the
alpha-1,3-GT gene through a genetic targeting event, a point
mutation was identified, which rendered the second allele
inactive.
[0142] Thus, in another aspect of the present invention, the
alpha-1,3-GT gene can be rendered inactive through at least one
point mutation. In one embodiment, one allele of the alpha-1,3-GT
gene can be rendered inactive through at least one point mutation.
In another embodiment, both alleles of the alpha-1,3-GT gene can be
rendered inactive through at least one point mutation. In one
embodiment, this point mutation can occur via a genetic targeting
event. In another embodiment, this point mutation can be naturally
occurring. In one specific embodiment the point mutation can be a
T-to-G mutation at the second base of exon 9 of the alpha-1,3-GT
gene (FIG. 2). Pigs carrying a naturally occurring point mutation
in the alpha-1,3-GT gene allow for the production of
alpha1,3GT-deficient pigs free of antibiotic-resistance genes and
thus have the potential to make a safer product for human use. In
other embodiments, at least two, at least three, at least four, at
least five, at least ten or at least twenty point mutations can
exist to render the alpha-1,3-GT gene inactive. In other
embodiments, pigs are provided in which both alleles of the
alpha-1,3-GT gene contain point mutations that prevent any
expression of functional alpha1,3GT. In a specific embodiment, pigs
are provided that contain the T-to-G mutation at the second base of
exon 9 in both alleles of the alpha-1,3-GT gene (FIG. 2).
[0143] Another aspect of the present invention provides a porcine
animal, in which both alleles of the alpha-1,3-GT gene are
inactivated, whereby one allele is inactivated by a genetic
targeting event and the other allele is inactivated via a naturally
occurring point mutation. In one embodiment, a porcine animal is
provided, in which both alleles of the alpha-1,3-GT gene are
inactivated, whereby one allele is inactivated by a genetic
targeting event and the other allele is inactivated due to presence
of a T-to-G point mutation at the second base of exon 9. In a
specific embodiment, a porcine animal is provided, in which both
alleles of the alpha-1,3-GT gene are inactivated, whereby one
allele is inactivated via a targeting construct directed to Exon 9
(FIG. 6) and the other allele is inactivated due to presence of a
T-to-G point mutation at the second base of exon 9.
[0144] V. Porcine Organs, Tissues, Cells and Cell Lines
[0145] The present invention provides, for the first time, viable
porcine in which both alleles of the alpha 1,3
galactosyltransferase gene have been inactivated. The invention
also provides organs, tissues, and cells derived from such porcine,
which are useful for xenotransplantation.
[0146] In one embodiment, the invention provides porcine organs,
tissues and/or purified or substantially pure cells or cell lines
obtained from pigs that lack any expression of functional
alpha1,3GT.
[0147] In one embodiment, the invention provides organs that are
useful for xenotransplantation. Any porcine organ can be used,
including, but not limited to: brain, heart, lungs, glands, brain,
eye, stomach, spleen, pancreas, kidneys, liver, intestines, uterus,
bladder, skin, hair, nails, ears, nose, mouth, lips, gums, teeth,
tongue, salivary glands, tonsils, pharynx, esophagus, large
intestine, small intestine, rectum, anus, pylorus, thyroid gland,
thymus gland, suprarenal capsule, bones, cartilage, tendons,
ligaments, skeletal muscles, smooth muscles, blood vessels, blood,
spinal cord, trachea, ureters, urethra, hypothalamus, pituitary,
adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands,
testes, seminal vesicles, penis, lymph, lymph nodes and lymph
vessels.
[0148] In another embodiment, the invention provides tissues that
are useful for xenotransplantation. Any porcine tissue can be used,
including, but not limited to: epithelium, connective tissue,
blood, bone, cartilage, muscle, nerve, adenoid, adipose, areolar,
bone, brown adipose, cancellous, muscle, cartaginous, cavernous,
chondroid, chromaffin, dartoic, elastic, epithelial, fatty,
fibrohyaline, fibrous, Gamgee, gelatinous, granulation,
gut-associated lymphoid, Haller's vascular, hard hemopoietic,
indifferent, interstitial, investing, islet, lymphatic, lymphoid,
mesenchymal, mesonephric, mucous connective, multilocular adipose,
myeloid, nasion soft, nephrogenic, nodal, osseous, osteogenic,
osteoid, periapical, reticular, retiform, rubber, skeletal muscle,
smooth muscle, and subcutaneous tissue.
[0149] In a further embodiment, the invention provides cells and
cell lines from porcine animals that lack expression of functional
alpha1,3GT. In one embodiment, these cells or cell lines can be
used for xenotransplantation. Cells from any porcine tissue or
organ can be used, including, but not limited to: epithelial cells,
fibroblast cells, neural cells, keratinocytes, hematopoietic cells,
melanocytes, chondrocytes, lymphocytes (B and T), macrophages,
monocytes, mononuclear cells, cardiac muscle cells, other muscle
cells, granulosa cells, cumulus cells, epidermal cells, endothelial
cells, Islets of Langerhans cells, pancreatic insulin secreting
cells, pancreatic alpha-2 cells, pancreatic beta cells, pancreatic
alpha-1 cells, blood cells, blood precursor cells, bone cells, bone
precursor cells, neuronal stem cells, primordial stem cells.,
hepatocytes, keratinocytes, umbilical vein endothelial cells,
aortic endothelial cells, microvascular endothelial cells,
fibroblasts, liver stellate cells, aortic smooth muscle cells,
cardiac myocytes, neurons, Kupffer cells, smooth muscle cells,
Schwann cells, and epithelial cells, erythrocytes, platelets,
neutrophils, lymphocytes, monocytes, eosinophils, basophils,
adipocytes, chondrocytes, pancreatic islet cells, thyroid cells,
parathyroid cells, parotid cells, tumor cells, glial cells,
astrocytes, red blood cells, white blood cells, macrophages,
epithelial cells, somatic cells, pituitary cells, adrenal cells,
hair cells, bladder cells, kidney cells, retinal cells, rod cells,
cone cells, heart cells, pacemaker cells, spleen cells, antigen
presenting cells, memory cells, T cells, B cells, plasma cells,
muscle cells, ovarian cells, uterine cells, prostate cells, vaginal
epithelial cells, sperm cells, testicular cells, germ cells, egg
cells, leydig cells, peritubular cells, sertoli cells, lutein
cells, cervical cells, endometrial cells, mammary cells, follicle
cells, mucous cells, ciliated cells, nonkeratinized epithelial
cells, keratinized epithelial cells, lung cells, goblet cells,
columnar epithelial cells, dopamiergic cells, squamous epithelial
cells, osteocytes, osteoblasts, osteoclasts, dopaminergic cells,
embryonic stem cells, fibroblasts and fetal fibroblasts. In a
specific embodiment, pancreatic cells, including, but not limited
to, Islets of Langerhans cells, insulin secreting cells, alpha-2
cells, beta cells, alpha-1 cells from pigs that lack expression of
functional alpha-1,3-GT are provided.
[0150] Nonviable derivatives include tissues stripped of viable
cells by enzymatic or chemical treatment these tissue derivatives
can be further processed via crosslinking or other chemical
treatments prior to use in transplantation. In a preferred
embodiment, the derivatives include extracelluar matrix derived
from a variety of tissues, including skin, urinary, bladder or
organ submucosal tissues. Also, tendons, joints and bones stripped
of viable tissue to include heart valves and other nonviable
tissues as medical devices are provided.
[0151] Therapeutic Uses
[0152] The cells can be administered into a host in order in a wide
variety of ways. Preferred modes of administration are parenteral,
intraperitoneal, intravenous, intradermal, epidural, intraspinal,
intrasternal, intra-articular, intra-synovial, intrathecal,
intra-arterial, intracardiac, intramuscular, intranasal,
subcutaneous, intraorbital, intracapsular, topical, transdermal
patch, via rectal, vaginal or urethral administration including via
suppository, percutaneous, nasal spray, surgical implant, internal
surgical paint, infusion pump, or via catheter. In one embodiment,
the agent and carrier are administered in a slow release
formulation such as a direct tissue injection or bolus, implant,
microparticle, microsphere, nanoparticle or nanosphere.
[0153] Disorders that can be treated by infusion of the disclosed
cells include, but are not limited to, diseases resulting from a
failure of a dysfunction of normal blood cell production and
maturation (i.e., aplastic anemia and hypoproliferative stem cell
disorders); neoplastic, malignant diseases in the hematopoietic
organs (e.g., leukemia and lymphomas); broad spectrum malignant
solid tumors of non-hematopoietic origin; autoimmune conditions;
and genetic disorders. Such disorders include, but are not limited
to diseases resulting from a failure or dysfunction of normal blood
cell production and maturation hyperproliferative stem cell
disorders, including aplastic anemia, pancytopenia,
agranulocytosis, thrombocytopenia, red cell aplasia,
Blackfan-Diamond syndrome, due to drugs, radiation, or infection,
idiopathic; hematopoietic malignancies including acute
lymphoblastic (lymphocytic) leukemia, chronic lymphocytic leukemia,
acute myelogenous leukemia, chronic myelogenous leukemia, acute
malignant myelosclerosis, multiple myeloma, polycythemia vera,
agnogenic myelometaplasia, Waldenstrom's macroglobulinemia,
Hodgkin's lymphoma, non-Hodgkin's lymphoma; immunosuppression in
patients with malignant, solid tumors including malignant melanoma,
carcinoma of the stomach, ovarian carcinoma, breast carcinoma,
small cell lung carcinoma, retinoblastoma, testicular carcinoma,
glioblastoma, rhabdomyosarcoma, neuroblastoma, Ewing's sarcoma,
lymphoma; autoimmune diseases including rheumatoid arthritis,
diabetes type I, chronic hepatitis, multiple sclerosis, systemic
lupus erythematosus; genetic (congenital) disorders including
anemias, familial aplastic, Fanconi's syndrome, dihydrofolate
reductase deficiencies, formamino transferase deficiency,
Lesch-Nyhan syndrome, congenital dyserythropoietic syndrome I-IV,
Chwachmann-Diamond syndrome, dihydrofolate reductase deficiencies,
formamino transferase deficiency, Lesch-Nyhan syndrome, congenital
spherocytosis, congenital elliptocytosis, congenital
stomatocytosis, congenital Rh null disease, paroxysmal nocturnal
hemoglobinuria, G6PD (glucose-6-phhosphate dehydrogenase) variants
1, 2, 3, pyruvate kinase deficiency, congenital erythropoietin
sensitivity, deficiency, sickle cell disease and trait, thalassemia
alpha, beta, gamma, met-hemoglobinemia, congenital disorders of
immunity, severe combined immunodeficiency disease (SCID), bare
lymphocyte syndrome, ionophore-responsive combined
immunodeficiency, combined immunodeficiency with a capping
abnormality, nucleoside phosphorylase deficiency, granulocyte actin
deficiency, infantile agranulocytosis, Gaucher's disease, adenosine
deaminase deficiency, Kostmann's syndrome, reticular dysgenesis,
congenital Leukocyte dysfunction syndromes; and others such as
osteoporosis, myelosclerosis, acquired hemolytic anemias, acquired
immunodeficiencies, infectious disorders causing primary or
secondary immunodeficiencies, bacterial infections (e.g.,
Brucellosis, Listerosis, tuberculosis, leprosy), parasitic
infections (e.g., malaria, Leishmaniasis), fungal infections,
disorders involving disproportionsin lymphoid cell sets and
impaired immune functions due to aging, phagocyte disorders,
Kostmann's agranulocytosis, chronic granulomatous disease,
Chediak-Higachi syndrome, neutrophil actin deficiency, neutrophil
membrane GP-180 deficiency, metabolic storage diseases,
mucopolysaccharidoses, mucolipidoses, miscellaneous disorders
involving immune mechanisms, Wiskott-Aldrich Syndrome, alpha
1-antirypsin deficiency, etc.
[0154] Diseases or pathologies include neurodegenerative diseases,
hepatodegenerative diseases, nephrodegenerative disease, spinal
cord injury, head trauma or surgery, viral infections that result
in tissue, organ, or gland degeneration, and the like. Such
neurodegenerative diseases include but are not limited to, AIDS
dementia complex; demyeliriating diseases, such as multiple
sclerosis and acute transferase myelitis; extrapyramidal and
cerebellar disorders, such as lesions of the ecorticospinal system;
disorders of the basal ganglia or cerebellar disorders;
hyperkinetic movement disorders, such as Huntington's Chorea and
senile chorea; drug-induced movement disorders, such as those
induced by drugs that block CNS dopamine receptors; hypokinetic
movement disorders, such as Parkinson's disease; progressive
supra-nucleo palsy; structural lesions of the cerebellum;
spinocerebellar degenerations, such as spinal ataxia, Friedreich's
ataxia, cerebellar cortical degenerations, multiple systems
degenerations (Mencel, Dejerine Thomas, Shi-Drager, and
Machado-Joseph), systermioc disorders, such as Rufsum's disease,
abetalipoprotemia, ataxia, telangiectasia; and mitochondrial
multi-system disorder; demyelinating core disorders, such as
multiple sclerosis, acute transverse myelitis; and disorders of the
motor unit, such as neurogenic muscular atrophies (anterior horn
cell degeneration, such as amyotrophic lateral sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy
body disease; Senile Demetia of Lewy body type; Parkinson's
Disease, Wernicke-Korsakoff syndrome; chronic alcoholism;
Creutzfeldt-Jakob disease; Subacute sclerosing panencephalitis
hallerrorden-Spatz disease; and Dementia pugilistica. See, e.g.,
Berkow et. al., (eds.) (1987), The Merck Manual, (15.sup.th) ed),
Merck and Co., Rahway, N.J.
[0155] The present invention is described in further detail in the
following examples. The examples provided below are intended to be
illustrative only, and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Production of Porcine Cells Heterozygous for the Alpha-1,3-GT
Gene
[0156] Isolation and Transfection of Primary Porcine Fetal
Fibroblasts
[0157] Fetal fibroblast cells (PCFF4-1 to PCFF4-10) were isolated
from 10 fetuses of the same pregnancy at day 33 of gestation. After
removing the head and viscera, fetuses were washed with Hanks'
balanced salt solution (HBSS; Gibco-BRL, Rockville, Md.), placed in
20 ml of HBSS, and diced with small surgical scissors. The tissue
was pelleted and resuspended in 50-ml tubes with 40 ml of DMEM and
100 U/ml collagenase (Gibco-BRL) per fetus. Tubes were incubated
for 40 min in a shaking water bath at 37.degree. C. The digested
tissue was allowed to settle for 3-4 min and the cell-rich
supernatant was transferred to a new 50-ml tube and pelleted. The
cells were then resuspended in 40 ml of DMEM containing 10% fetal
calf serum (FCS), 1.times. nonessential amino acids, 1 mM sodium
pyruvate and 2 ng/ml bFGF, and seeded into 10 cm. dishes. All cells
were cryopreserved upon reaching confluence. SLA-1 to SLA-10 cells
were isolated from 10 fetuses at day 28 of pregnancy. Fetuses were
mashed through a 60-mesh metal screen using curved surgical forceps
slowly so as not to generate excessive heat. The cell suspension
was then pelleted and resuspended in 30 ml of DMEM containing 10%
FCS, 1.times. nonessential amino acids, 2 ng/ml bFGF, and 10
.mu.g/ml gentamycin. Cells were seeded in 10-cm dishes, cultured
one to three days, and cryopreserved. For transfections, 10 .mu.g
of linearized vector DNA was introduced into 2 million cells by
electroporation. Forty-eight hours after transfection, the
transfected cells were seeded into 48-well plates at a density of
2,000 cells per well and were selected with 250 .mu.g/ml of
G418.
[0158] Knockout Vector Construction
[0159] Two alpha-1,3-GT knockout vectors, pPL654 and pPL657, were
constructed from isogenic DNA of two primary porcine fetal
fibroblasts, SLA1-10 and PCFF4-2 cells. A 6.8-kb alpha-1,3-GT
genomic fragment, which includes most of intron 8 and exon 9, was
generated by PCR from purified DNA of SLAT-10 cells and PCFF4-2
cells, respectively. The unique EcoRV site at the 5' end of exon 9
was converted into a SalI site and a 1.8-kb IRES-neo-poly A
fragment was inserted into the SalI site. IRES (internal ribosome
entry site) functions as a translation initial site for neo
protein. Thus, both vectors have a 4.9-kb 5' recombination arm and
a 1.9-kb 3' recombination arm (FIG. 6).
[0160] 3'PCR and Long-Range PCR
[0161] Approximately 1,000 cells were resuspended in 5 .mu.l embryo
lysis buffer (ELB) (40 mM Tris, pH 8.9, 0.9% Triton X-100, 0.9%
NP40, 0.4 mg/ml Proteinase K), incubated at 65.degree. C. for 15
min to lyse the cells and heated to 95.degree. C. for 10 min to
inactivate the Proteinase K. For 3' PCR analysis, fragments were
amplified using the Expand High Fidelity PCR system (Roche
Molecular Biochemicals) in 25 .mu.l reaction volume with the
following parameters: 35 cycles of 1 min at 94.degree. C., 1 min at
60.degree. C., and 2 min at 72.degree. C. For LR-PCR, fragments
were amplified by using TAKARA LA system (Panvera/Takara) in 50
.mu.l reaction volume with the following parameters: 30 cycles of
10 s at 94.degree. C., 30 s at 65.degree. C., 10 min+20 s
increase/cycle at 68.degree. C., followed by one final cycle of 7
min at 68.degree. C. 3'PCR and LR-PCR conditions for purified DNA
was same as cells except that 1 of purified DNA (30 .mu.g/ml) was
mixed with 4 .mu.l ELB.
[0162] Southern blot analysis of cell samples Approximately 106
cells were lysed overnight at 60.degree. C. in lysis buffer (10 mM
Tris, pH 7.5, 10 mM EDTA, 10 mM NaCl, 0.5% (w/v) Sarcosyl, 1 mg/ml
proteinase K) and the DNA precipitated with ethanol. The DNA was
then digested with BstEII and separated on a 1% agarose gel. After
electrophoresis, the DNA was transferred to a nylon membrane and
probed with the 3'-end digoxigenin-labeled probe. Bands were
detected using a chemiluminescent substrate system (Roche Molecular
Biochemicals).
[0163] Results Antibiotic (G418) resistant colonies were screened
by 3' PCR with neo442S and .alpha.GTE9A2 as forward and reverse
primers. Neo442S is at the 3' end of the neo gene and .alpha.GTE9A2
is at the 3' end of exon 9 in sequences located outside of the 3'
recombination arm (FIG. 6). Therefore, only through successful
targeting at the .alpha.1,3GT locus would the expected 2.4 kb PCR
product be obtained. From a total of seven transfections in four
different cell lines, 1105 G418 resistant colonies were picked, of
which 100 (9%) were positive for .alpha.1,3 GT gene disruption in
the initial 3' PCR screen (range 2.5-12%). Colonies 657A-A8,
657A-I6, and 657A-I11 showed the expected 2.4 kb band, while
control PCFF4-6 cells, and another G418 resistant colony, 657A-P6,
were negative. A portion of each 3' PCR positive colony was frozen
down immediately, in several small aliquots, for future use in NT
experiments, while the rest of cells were expanded for long-range
PCR (LR-PCR) and Southern analysis.
[0164] Since PCR analysis to detect recombination junctions, or
mRNA analysis (RT-PCR) can generate false positive results, a
long-range PCR, which would encompass the entire targeted region,
was performed. The LR-PCR covers the 7.4 kb .alpha.1,3GT genomic
sequence from exon 8 to the end of exon 9, with both primers
(.alpha.GTE8S and .alpha.GTE9A2) located outside of the
recombination region (FIG. 2). The control PCFF4-6 cells, and the
3' PCR-negative colony, 657A-P6, showed only the endogenous 7.4 kb
band from the wild-type .alpha.1,3GT locus. In contrast, three of
the 3' PCR positive colonies, 657A-A8, 657A-I6 and 657A-I11, showed
both the 7.4 kb endogenous band, and a new 9.2 kb band, of the size
expected for targeted insertion of the 1.8 kb IRES-neo cassette
into the .alpha.1,3GT locus.
[0165] Approximately half (17/30) of the LR-PCR positive colonies
were successfully expanded to yield sufficient cell numbers
(1.times.106 cells) for Southern analysis. It was anticipated that
the colonies would be heterozygous for knockout at the .alpha.1,3
GT locus, and thus they should have one normal, unmodified gene
copy, and one disrupted copy of the .alpha.1,3 GT gene. With BstEII
digestion, the .alpha.1,3 GT knockout cells should show two bands:
one 7 kb band of the size expected for the endogenous .alpha.1,3 GT
allele, and a 9 kb band characteristic of insertion of the IRES-neo
sequences at the .alpha.1,3 GT locus (FIG. 2). All 17 LR-PCR
positive colonies were confirmed by Southern analysis for the
knockout. The same membranes were re-probed with sequences specific
for neo and the 9 kb band was detected with the neo probe, thus
confirming targeted insertion of the IRES-neo cassette at the
disrupted .alpha.1,3GT locus.
Example 2
Production of Porcine Cells Homozygous for the Alpha-1,3-GT
Gene
[0166] Heterozygous alpha-1,3-GT knockout fetal fibroblasts,
(657A-I11 1-6) cells, were isolated from a day-32 pregnancy as
described above (See also Dai et al. Nature Biotechnology 20:451
(2002)). An ATG (start codon)-targeting alpha-1,3-GT knockout
vector was constructed (pPL680), which also contained a neo gene,
to knock out the second allele of the alpha-1,3-GT gene. These
cells were transfected by electroporation with pPL680 and selected
for the alpha1,3Gal-negative phenotype with purified C. difficile
toxin A (described below).
Example 3
Selection with C. difficile Toxin a for Porcine Cells Homozygous
for the Alpha-1,3-GT Gene
[0167] Toxin A Cyototoxicily Curve
[0168] Porcine cells (PCFF4-6) were exposed for 1 hour or overnight
to ten-fold serial dilutions of toxin A (0.00001 .mu.g/ml to 10
.mu.g/ml). Cells were cultured in 24 well plates and were incubated
with the toxin for 1 hour or overnight at 37 C. The results of this
exposure are detailed in Table 2. Clearly, a 1 hour exposure to
toxin A at >1 .mu.g/ml resulted in a cytotoxic effect on >90%
of the cells. A concentration of toxin A at or slightly above 1
.mu.g/ml therefore was chosen for selection of genetically altered
cells.
TABLE-US-00003 TABLE 2 Toxin A toxicity at 1 hour and overnight
exposure [Toxin A], .mu.g/ml 1 hour incubation Overnight incubation
0 100% confluency 100% confluency .00001 100% confluency 100%
confluency .0001 100% confluency 100% confluency .001 100%
confluency 100% confluency .01 100% confluency 50% confluency, 50%
rounded .1 90% confluency Same as 10 ug/ml 1 >90% rounded Same
as 10 ug/ml 10 All cells rounded up All cells rounded up, some
lifted
[0169] Disaggregated cells from a porcine embryo (1-11:1-6) which
contained a previously identified targeted knockout in one allele
of the gal alpha-1,3-GT gene (Dai et al.) were transfected with 10
ug linearized vector DNA (promoter trap) by electroporation. After
48 hours, the cells were seeded into 48 well plates at a density of
2000 cells per well and selected with 250 ug/ml G418. Five days
post-transfection, media was withdrawn from the wells, and replaced
with 2 ug/ml toxin A in culture media (DMEM high glucose with 2.8
ng/ml bFGF and 20% FCS). Cells were exposed to the selective effect
of toxin A for 2 hours at 37 C. The toxin A-containing media, along
with any affected cells that have released from the plate surface,
was withdrawn, the remaining cells washed with fresh media, and the
media without toxin A replaced. Ten days later, cells were again
exposed to toxin A at 1.3 ug/ml in media for 2 hours at 37 C. The
media, toxin A, and any cells in solution were removed, the
remaining cells washed, and the media replaced.
[0170] Sixteen days post-transfection, a single colony that
exhibited toxin A insensitivity, designated 680B1, was harvested
and a portion sent for DNA analysis and lectin staining. DNA
analysis indicated that the toxin A insensitivity was not due to
integration of the second target vector; however, the cells did not
stain with GSL IB-4 lectin, indicating that a functional knockout
of the locus had occurred. The 680B1 double knockout cells were
used for nuclear transfer into 5 recipients and three pregnancies
resulted. Two of these pregnancies spontaneously aborted in the
first month; the four fetuses from the remaining pregnancy were
harvested on day 39 of the pregnancy and the cells disaggregated
and seeded into tissue culture. These fetal cells (680B1-1,
680B1-2, 680B1-3, 680B1-4) were exposed to toxin A at 1 ug/ml for 1
hour at 37 C, followed by medium removal, cell washing, and medium
replacement without toxin A. Fetuses 1,2, and 4 were not affected
by toxin A, whereas most of the cells from fetus 3 rounded up,
indicating that this embryo was sensitive to the cytotoxic effects
of the toxin A.
[0171] Fetuses 1,2, and 4 did not bind GS IB4 lectin, as indicated
by FACS analysis (see Table 3), while fetus 3 did bind lectin. This
suggests that fetuses 1, 2, and 4 do not carry the epitope alpha
1,3 gal for which this particular lectin is specific.
TABLE-US-00004 TABLE 3 FACS Results of 680B1-1 to 680B1-4 Cells
with GS-IB4 Lectin GS IB4 lectin positive cells (%) 50 .mu.g/ml 100
.mu.g/ml Cell Unstaining IB4 lectin IB4 lectin HeLa Cells (Negative
CTL) .sup. 1% 2% 2.8% PCFF4-6 cells (Positive CTL) 0.2% 76% 91%
PFF4 cells (Positive CTL) 1.5% 82% 94% 680B 1-1 cells 0.6% 0.8%
0.9% 680B 1-2 cells 1.2% 1.2% 1.1% 680B1-3 cells .sup. 8% 35% 62%
680B1-4 cells 0.6% 0.8% 0.9%
[0172] A complement fixation assay was run on cells from all four
fetuses. The complement lysis assay was developed as a bioassay for
lack of alpha gal expression. Human serum contains high levels of
pre-formed antibody against alpha gal as well as the full portfolio
of complement regulatory proteins (the C3 pathway). The presence of
alpha gal on the surface of a cell, upon binding of anti-alpha gal
antibody, activates the complement cascade, and results in
complement-mediated cell lysis. Alpha-gal negative cells would be
resistant to complement mediated lysis. In three separate tests, B1
and control pig cells were exposed to human serum plus complement,
and assays performed to evaluate sensitivity or resistance to
alpha-gal-initiated, complement-mediated cell lysis. The assay was
performed with B1-1, B1-2, and B1-4 cells, as well as heterozygous
GT KO cells (B1-3, gal positive), and with wild-type alpha-gal (+)
PCFF4-6 pig cells as a control. Cells were exposed to one of three
treatments; two negative controls, bovine serum albumin (BSA), and
heat-inactivated human serum (HIA-HS) do not contain any functional
complement protein and thus would not be expected to cause any
significant cell lysis; the third treatment, non-heat-inactivated
human serum (NHS) contains functional human complement as well as
anti-gal specific antibodies, and thus would be expected to lyse
cells which have galactose alpha 1,3 galactose on their cell
surface.
[0173] The results shown in FIG. 1 clearly demonstrate that B1-1,
B-2 and B1-4 cells are resistant to human complement-mediated lysis
while B1-3 cells, which is .alpha.1,3 Gal positive, is still as
sensitive to human plasma as are wild-type PCFF4-6 cells.
[0174] Sequencing results of cDNA from all fetuses indicated that
fetuses 1,2 and 4 contain a point mutation in the second alpha 1,3
GT allele, a change that could yield a dysfunctional enzyme (see
FIG. 2). This mutation occurred at bp424 of the coding region,
specifically, the second base pair of exon 9, of the alpha-1,3-GT
(GGTA1) gene (GenBank Accession No. L36152) as a conversion of a
thymine to a guanine residue, which results in an amino acid
substitution of tyrosine at aa 142 to an aspartic acid.
[0175] This is a significant conversion, as the tyrosine, a
hydrophilic amino acid, is a critical component of the UDP binding
site of alpha 1,3GT (see FIG. 3). Analysis of the crystal structure
of bovine alpha-1,3-GT protein showed that this tyrosine is the
center of the catalytic domain of the enzyme, and is involved in
UDP-Gal binding (Gastinel et. al., EMBO Journal 20(4): 638-649,
2001). Therefore, a change from tyrosine (a hydrophobic amino acid)
to aspartic acid (a hydrophilic amino acid) would be expected to
cause disruption of the .alpha.GT function (as observed).
[0176] To confirm that the mutated cDNA will not make functional
.alpha.GT protein., the cDNAs from the second allele of all 4 cells
were cloned into an expression vector and this GT expression vector
transfected into human fibroblast cells (HeLa cells) as well as
into primary Rhesus monkey cells. As humans and Old World monkeys
lack a functional alpha 1,3 GT gene, the HeLa cells would not have
an alpha 1,3 galactose on their cell surface (as assayed by lectin
binding experiments). Results showed that the HeLa and monkey
cells, when transfected with cDNA obtained from B1-1, B1-2 and B1-4
cells, were still .alpha.1,3 Gal negative by IB4-lectin staining,
while Hela and Rhesus monkey cells transfected with cDNA from the
B1-3, made a functional alpha 1,3 GT transcript and subsequently
were .alpha.1,3Gal positive. Clearly, cells with the aspartate
mutation (instead of tyrosine) cannot make functional alpha 1,3
galactosyl transferase
Example 4
Generation of Cloned Pigs Using Homozygous Alpha 1,3 GT-Deficient
Fetal Fibroblasts as Nuclear Donors
[0177] Preparation of cells for nuclear transfer. Donor cells were
genetically manipulated to produce cells homozygous for alpha 1,3
GT deficiency as described generally above. Nuclear transfer was
performed by methods that are well known in the art (see, e.g., Dai
et al., Nature Biotechnology 20: 251-255, 2002; and Polejaeva et
al., Nature 407:86-90, 2000), using toxin A-selected porcine
fibroblasts as nuclear donors that were produced as described in
detail hereinabove
[0178] Embryo transfers and resulting live births. In the initial
attempt to produce live alpha-1,3-GT dKO pigs by nuclear transfer,
a total of 16 embryo transfers were performed with genetically
manipulated donor cells. Nine initial pregnancies were established
but only two went beyond Day 75 of gestation. Five piglets were
born on the 25, Jul. 2002. One piglet died immediately after birth
and another four were born alive and appeared normal (FIG. 4).
Example 5
Analysis of Homozygous Alpha 1,3 GT Knockout Pigs
[0179] Tail fibroblast cells and umbilicus tissue sections were
obtained from all 5 double knockout piglets and stained using the
GS-IB4 lectin as described previously. No staining was observed,
indicating a complete lack of galactose alpha 1,3 galactose epitope
on the surface of tissues from these animals (data not shown).
Aorta endothelial cells and muscle and tail fibroblasts isolated
from the dead piglet (761-1) were negative with GS-IB4 lectin
staining. FACS analysis of muscle fibroblasts from piglet 761-1
also showed a negative result for GS-IB4 binding. Tissue sections
of liver, kidney, spleen, skin, intestine, muscle, brain, heart,
pancreas, lung, aorta, tongue, umbilicus, and tail obtained from
piglet 761-1 were all negative with GS-IB4 staining, indicating a
complete lack of detectable cell surface alpha 1,3Gal epitopes
(Phelps et al., Science 299: 411-414, 2003 including figure
S3).
[0180] We performed an in vivo immunogenicity test with alpha
1,3GT-knockout mice. We injected islet-like cell clusters (ICCs)
isolated from the pancreas of piglet 761-1 intraperitoneally into
alpha 1,3GT knockout mice. We used ICCs from a neonatal wild-type
piglet as a control. As shown in FIG. 5, no increase in the titer
of immunoglobulin M (IgM) to alpha 1,3Gal was observed in alpha
1,3GT knockout mice after injection with ICCs from the alpha 1,3GT
DKO piglet, in contrast to significant IgM titer increases observed
in those mice injected with wild-type piglet ICCs (Phelps et al.,
Science 299: 411-414, 2003 including figure S4). This result
clearly demonstrates that the DKO piglet cells do not make any
alpha 1,3Gal epitopes.
[0181] Sequencing of DNA obtained from all five piglets confirmed
the presence of the mutation at by 424 of the GGTA1 gene, as
observed in the 680B1-2 cells used to clone these animals (FIG.
2).
[0182] Since this first successful production of a litter of
alpha-GT dKO pigs, two subsequent litters of dKO piglets have been
produced by nuclear transfer, in one case (litter 662) using the
dKO fetal fibroblasts as nuclear donor cells. Litter 660 was
produced by nuclear transfer using tail fibroblast cells from a
member of the litter 761 as nuclear donor. These births are
summarized in Table 4.
TABLE-US-00005 TABLE 4 Summary of alpha-GT double knockout births
produced by nuclear transfer Litter ID Nuclear Donor No. Births
Live Births 761 680B: 1-2 5 4 662 680B: 1-2 1 0 660 761-5 4 2
Example 6
Breeding of Heterozygous Alpha 1,3 GT Single Knockout (SKO) Male
and Female Pigs to Establish a Miniherd of Double Knockout (DKO)
Pigs
[0183] A total of 29 Southern blot confirmed cloned GT-SKO females
and 25 Southern blot confirmed GT-SKO male cloned pigs have been
generated to date. These male and female heterozygous (single gene
alpha1,3GT knockout pigs) have been bred by natural breeding and by
artificial insemination(AI), in order to generate a herd of DKO
pigs for use in preclinical studies and human clinical trials. We
have produced 16 alpha1,3-GT DKO piglets from 13 litters.
[0184] This invention has been described with reference to
illustrative embodiments. Other embodiments of the general
invention described herein and modifications there of will be
apparent to those of skill in the art and are all considered within
the scope of the invention.
Sequence CWU 1
1
415PRTArtificial SequenceExon 9 wild type amino acid sequence 1Tyr
Ile Glu His Tyr1 5227DNAArtificial SequenceExon 8-9 wild type DNA
sequence 2gctgtcggaa gatacattga gcattac 2735PRTArtificial
SequenceExon 9 mutated amino acid sequence 3Asp Ile Glu His Tyr1
5427DNAArtificial SequenceExon 8-9 mutated DNA sequence 4gctgtcggaa
gagacattga gcattac 27
* * * * *
References