Note: Descriptions are shown in the official language in which they were submitted.
~ W092/22~6 ~ PCT/US92/05~
--1--
PRODUCTION OF HUMAN HEMOGLOBIN IN TRANSGENIC PIGS
1. INTRODUCTION
The present invention relates to the use of
transgenic pigs for t~e production of human
hemoglobin. The transgenic pigs of the invention may
be used as an efficient and economical source of cell-
free human hemoglobin that may be used for
transfusions and other medical applications in humans.
2. BACKGROUND OF THE INVENTION
2.1. HEMQGLOBIN
Oxygen absorbed through the lungs is carried
by hemoglobin in red blood cells for delivery to
tissues throughout the body. At high oxygen tensions,
such as those found in the proximity of the lungs,
oxygen binds to hemoglobin, but is released in areas
of low cxygen tension, where it is needed.
Each hemoglobin molecule consists of two
alpha globin and two beta globin subunits. Each
subunit, in turn, is noncovalently associated with an
iron-containing heme group capable of carrying an
oxygen molecule. Thus, each hemoglobin tetramer is
capable of binding four molecules of oxygen. The
subunits work together in switching between two
conformational states to facilitate uptake and release
of oxygen at the lungs and tissues, respectively.
This effect is commonly referred to as heme-heme
interaction or cooperativity.
The hemoglobins of many animals are able to
interact with biologic effector molecules that can
further enhance oxygen binding and release. This
enhancement is manifested in changes which affect the
allosteric equilibrium between the two conformational
~ 1 l ~ c ~ ~
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states of hemoglobin. For example, human and pig
hemoglobin can bind 2, 3 diphosphoglycerate (2,3 DPG),
which influences the equilibrium between the two
conformational states of the tetramer and has the net
effect of lowering the overall affinity for oxygen at
the tissue level. As~a result, 2, 3-DPG increases the
efficiency of oxygen delivery to the tissues.
~:~ 10 2.2. GLOBIN GENE EXPRESSION
Hemoglobin protein is expressed in a tissue
specific manner in red blood cells where it accounts
for approximately ninety percent of total cellular
~; protein. Thus, red blood cells, which have lost their
nucleus and all but a minimal number of organelles,
are effectively membrane-enclosed packets of
- ~ ~ hemoglobin dedicated to oxygen transfer.
Humans and various other species produce
"
- different types of hemoglobin during embryonic, fetal,
; 20 and adult developmental periods. Therefore, the
;~ factors that influence globin gene expression must be
able to achieve tissue specific control, quantitative
; control, and developmentally regulated control of
globin expression.
Human globin genes are found in clusters on
chromosome 16 for alpha (~) globin and chromosome 11
for beta (~) globin. The human beta globin gene
cluster consists of about 50 kb of DNA that includes
one embryonic gene encoding epsilon (~) globin, two
fetal genes encoding gamma (~) G and gamma A globin,
and two adult genes encoding delta (~) and beta (~)
globin, in that order (Fritsch-~t al., 1980, Cell
9:959-972)-
It has been found that DNA sequences bothupstream and downstream of the ~ globin translation
initiation site are involved in the regulation of ~
,,
~ W092/22646 2 1 1 1 3 1 8 PCT/US92/~
globin gene expression (Wright et al., 1984, Cell
38:263). In particular, a series of four Dnase I
super hypersensitive sites (now referred to as the
locus control region, or LCR) located about 50
kilobases upstream of the human beta globin gene are
extremely important in=eliciting properly regulated
beta globin-locus expression (Tuan et al., 1985, Proc.
Natl. Acad. Sci. U.S.A. 83:1359-1363; PCT Patent
lo Application WO 8901517 by Grosveld; Behringer et al.,
1989, Science 245:971-973, Enver et al., 1989, Proc.
Natl. Acad. Sci. U.S.~. 86:7033-7037; Hanscombe et
al., 1989, Genes Dev. 3:1572-1581; Van Assendelft et
al., 1989, Cell 56:967-977; ~rosveld et al;, 1987,
Cell 51:975-985).
2.3. THE NEED FOR A BLOOD SUBSTITUTE
Recently, the molecular aspects of globin
gene expression have met with even greater interest as
researchers have attempted to use genetic engineering
to produce a synthetic blood that would avoid the
pitfalls of donor generated blood. In 1988, between
12 million and 14 million units of blood were used in
the United States alone (Andrews, February 18, 1~90,
New York Times), an enormous volume precariously
dependent on volunteer blood donations. About 5
percent of donated blood is infected by hepatitis
virus (Id.) and, although screening procedures for HIV
infection are generally effective, the prospect of
contracting transfusion related A.I.D.S. remains a
much feared possibility. Furthermore, transfused
blood must be compatible with-~he blood type of the
transfusion recipient; the donated blood supply may be
unable to provide transfusions to individuals with
3j rare blood types. In contrast, hemoglobin produced by
genetic engineering would not require blood type
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W092/22~6 PCT/US92/0
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matching, would be virus-free, and would be available
in potentially unlimited amounts. Several research
groups have explored the possibility of expressing
hemoglobin in microorganisms- For example, see
International Application No. PCT/US88/01534 by
Hoffman and Nagai, wh-l~h presents, in working
examples, production of human globin protein in E.
coli.
2.4. TRANSGENIC ANIMALS
A transgenic animal is a non-human animal
containing at least one foreign gene, called a
transgene, in its genetic ma~erial. Preferably, the
transgene is contained in the animal's germ line such
that it can be transmitted to the animal's offspring.
A number of techniques may be used to introduce the
transgene into an animal's genetic material,
including, but not limited to, microinjection of the
transgene into pronuclei of fertilized eggs and
manipulation of embryonic stem cells (U.S. Patent No.
4,873,191 by Wagner and Hoppe; Palmiter and Brinster,
1986, Ann. Rev. Genet. 20:465-499; French Patent
Application 2593827 published August 7, 1987).
Transgenic animals may carry the transgene in all
their cells or may be genetically mosaic.
Although the majority of studies have
involved transgenic mice, other species of transgenic
animal have also been produced, such as rabbits,
sheep, pigs (Hammer et al., 1985, Nature 315:680-683)
and chickens (Salter et al., 1987, Virology 157:236_
240). Transgenic animals are~c~rrently being
developed to serve as bioreactors for the production
of useful pharmaceutical compounds (Van Brunt, 1988,
Bio/Technology 6:1149-1154; Wilmut et al., 1988, New
Scientist (July 7 issue) pp. 56-59).
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Methods of expressing recombinant protein
via transgenic livestock have an important theoretical
advantage over protein production in recombinant
bacteria and yeast; namely, the ability to produce
large, complex proteins in which post-translational
modifications, inclùd~g glycosylation,
phosphorylation, subunit assembly, etc. are critical
for the activity of the molecule.
In practice, however, the creation of
transgenic livestock has proved problematic. Not only
is it technically difficult to produce transgenic
embryos, but mature transgenic animals that produce
significant quantities of re~ombinant protein may
prove inviable. In pigs in particular, the experience
has been that pigs carrying a growth hormone encoding
transgene (the only transgene introduced into pigs
` prior to the present invention) suffered from a numberof health problems, including severe arthritis, lack
of coordination in their rear legs, susceptibility to
stress, anoestrus in gilts and lack of libido in boars
(Wilmut et al., sura). This is in contrast to
transgenic mice carrying a growth hormone transgene,
- which appeared to be healthy (Palmiter et al., 1982,
Nature 300:611-615). Thus, prior to the present
invention, healthy transgenic pigs (which efficiently
express their transgene(s3) had not been produced.
2.5. EXPRESSION OF GLOBIN GENES IN TRANSGENIC ANIMALS
Transgenic mice carrying human globin
~ransgenes have been used in studying the molecular
biology of globin gene express~on. A hybrid
mouse/human adult beta globin gene was described by
Magram et al. in 1985 (Nature 315:338-340). Kollias
et al. then reported regulated expression of human
gamma-A, beta, and hybrid betatgamma globin genes in
,~,~ ..
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wo92t22 ~ PCT/US92/OS~
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transgenic mice (1986, Cell 46:89-94). Transgenic
mice expressing human fetal gamma globin were studied
by Enver et al. (1989, Proc. Natl. Acad. Sci. U.S.A.
86:7033-7037) and Constantoulakis et al. (1991, Blood
77:1326-1333). Autonomous developmental control of
human embryonic globin~-gene switching in transgenic
mice was observed by Raich et al. (1990, Science
250:11~7-1149).
Transgenic mouse models for a variety of
disorders of hemoglobin or hemoglobin expression have
been developed, including sickle cell disease (Rubin
et al., 1988, Am. J. Human Genet. 42:585-591; Greaves
et al., 1990, Nature 343:183~185; Ryan et al., 1990,
Science 247:566-568; Rubin et al., 1991, J. Clin.
Invest. 87:639-647); thalassemia (Anderson et al.,
1985, Ann. New York Acad. Sci. (USA) 445:445-451;
~ ~ Sorenson et al., 1990, Blood 75:1333-1336); and
- hereditary persistence of fetal hemoglobin (Tanaka et
' 20 al., 19~0, Ann. New York Acad. Sci. (USA~ 612:167-
- 178).
~::
Concurrent expression of human alpha and
beta globin has led to the production of human
hemoglobin in transgenic mice (Behringer et al., 1989,
Science 245:971-973; Townes et al., 1989, Prog. Clin.
Biol. Res. 316A:47-61; Hanscombe et al., 1989, Genes
Dev. 3:1572-1581). It was observed by Hanscomke et
al. (supra) that transgenic fetuses with high copy
numbers of a transgene encoding alpha but not beta
globin exhibited severe anemia and died prior to
, blirth. Using a construct with both human alpha and
beta globin genes under the control of the beta globin
LCR, live mice with low copy numbers were obtained
(Id.). Metabolic labeling experiments showed balanced
mouse globin synthesis, but imbalanced human globin
.
,~
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~,W092/22~6 2 1 ~ ~ 3 ~ 8 PCT/US92/05~
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synthesis, with an alpha/beta biosynthetic ratio of
about 0.6 (Id.).
3. Sl)MMARY OF THE INVENTION
The present invention relates to the use~of
transgenic pigs for t~e production of human hemoglobin
and/or human globin. It is based, at least in part,
on the discovery that transgenic pigs may be generated
that express human hemoglobin in their erythrocytes
and are healthy, suffering no deleterious effects as a
result of heterologous hemoglobin production.
In particular embodiments, the present
invention provides for transgenic pigs that express
human globin genes. Such animals may be used as a
particularly efficient and economical source of human
hemoglobin, in light of (i) the rela~ively short
periods of gestation and sexual maturation in pigs;
(ii) the size and frequency of litters, (iii~ the
relatively large size of the pig which provides
proportionately large yields of hemoglobin; and ( iY)
functional similarities between pig and human
hemoglobins in the regulation of oxygen binding
affinity which enables the transgenic pigs to remain
healthy in the presence of high levels of human
hemoglobin.
The present invention also provides for
recombinant nucleic acid constructs that may be used
to generate transgenic pigs. In preferred
embodiments, such constructs place the human alpha and
beta globin genes under the same promoter so as to
avoid deleterious effects of g`~obin chain imbalance
and/or titration of transcription factors due to
constitutive ~-globin promoter activity in an
inappropriate cell type (e.g. a primitive
erythrocyte).
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W092~22~ PCT/US92/OS~
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In an additional embodiment, the present
invention provides for a hybrid hemoglobin that
comprises human ~ globin and pig ~ globin. The whole
blood from transgenic pigs expressing this hybrid
hemoglobin appears to exhibit a Ps(, that is
advantageously higherrthan that of native human or pig
blood.
The present invention also provides for a
lo method of producing human hemoglobin comprising (i)
introducing a human alpha globin and a human beta
globin gene, under the control of a suitable promoter
or promoters, into the genetic material of a pig so as
to create a transgenic pig that expresses human
lS hemoglobin in at least some of its red blood cells;
(ii) collecting red blood cells from the transgenic
pig; (iii) releasing the contents of the collected red
blood cells; and (iv) subjecting the released contents
~- of the red blood cells to a purification procedure
that substantially separates human hemoglobin from pig
hemoglobin. In a preferred embodiment of the
invention, human hemoglobin may be separated from pig
hemoglobin by DEAE anion exchange column
chromatography.
4. DESCRIPTION OF THE FIGURES
Figure 1. Recombinant nucleic acid constructs.
A. Construct ~ (the "116 construct); B.
Construct ap~ (the "185" construct); C. Construct
~p~ (the "290" construct); D. Construct ~p~; E.
Construct ~p~ap~; F. Construct ~p~ carrying a
~108 Asn -> Asp mutation ~the "hemoglobin
Yoshizuka construct"); G. Construct p~ carrying
a ~108 Asn -> Lys mutation (the "hemoglobin
Presbyterian construct"~; H. Construct ~p~
coinjected with LCR ~ (the "285" construct); I.
;~ .
~ W092/22~6 2 1 1 ~ 3 4 ~ PCT/US92/05~
_ g
Construct ap~ carrying an ~134 Thr -~ Cys
mutation (the "227" construct); J. Construct ~p~
carrying an ~104 Cys-> Ser mutation (the "227"
construct), a ~93 Cys -> Ala mutation, and a ~112
Cys -> Val mutation (the "228" construct); K.-
Construct ~p~ (t~e "263" construct~; and L.
Construct ~p~ ) coinjected with LCR ~ (the
"274" construct); M. Construct LCR ~ coinjected
with LCR ~ (the "240" construct); N. Construct
~p~ carrying a ~61 Lys -> Met mutation (the
"Hemoglobin Boloqna" construct); 0. Construct LCR
(the "318" construct); P. Construct LCR ~
(the "319" construct); ~. Construct LCR ~ (the
"329" construct); R. Construct LCR ~(Pi~p)~ (the
"339" construct); S. Construct ~p~ carrying an
-~ ~75 Asp -> Cys mutation (the "340" construct3; T.
Construct ap~ carrying an ~42 Tyr -> Arg mutation
(the "341" construct); U. Construct LCR ~a (the
20~ "343" construct); V. Construct LCR ~ (the "347"
construct); W. Construct ~p~ carrying an ~42 Tyr
-> Lys mutation; X. Construct ap~ carrying an ~42
Tyr -> Arg mutation; and a ~99 Asp -> Glu
mutation; Y. Construct ~p~ carrying an ~42 Tyr -~
Lys mutation; and a ~99 Ai~p -~ Glu mutation.
Figure 2. Transgenic pig.
Figure 3. Demonstration of human hemoglobin
expression in transgenic pigs. A. Isoelectric
focusing gel analysis. B. Triton-acid urea gel
of hemolysates of red blood cells representing
human blood (lane 1); blood from transgenic pig
12-1 (lane 2), 9-3 (lane ~t, and 6-3 (lane 4);
and pig blood (lane 5) shows under-expression of
human ~ globin relative to human ~ globin in the
transgenic animals.
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W092/22~6 PCT~US92/05~
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Figure 4. Separation of human hemoglobin and pig
hemoglobin by DEAE chromatography. A. Hemolyzed
mixture of human and pig red blood cells; B.
S Hemolysate of red blood cells collected from
transgenic pig 6-3. C. Human and mouse
hemoglobin do not separate by DEAE chromatography
under these conditions. D. Isoelectric focusing
of human hemoglobin purified from pig hemoglobin.
Figure 5. Isoelectric focussing gel of reassociated
pig hemoglobin (lane 1); reassociated pig/human
hemoqlobin mixture (lanes 2 and ~); reassociated
-~ human hemoglobin (lane 3); and transgenic pig
hemoglobin (lane 5). ~ ~
Figure 6. Separation of human hemoglobin by QCIP
chromatography.
Figure 7. Oxygen affinity of transgenic hemoglobin.
5. DETAILED DESCRIPTION OF THE INVENTION
The present invention provides for a method
of producing human hemoglobin that utilizes transgenic
pigs, novel globin-encoding nucleic acid constructs,
and transgenic pigs that express human hemoglobin.
For purposes of clarity of description, and not by way
of limitation, the detailed description of the
invention is divided into the following subsections~ ;r
(i) preparation of globin gene constructs;
(ii) preparation of transgenic pigs;
(iii) preparation of human hemoglobin and
its separation from pig hemoglobin;
and
tiv) preparation o~~human/pig hybrid
hemoglobin.
, . . .
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~ jWO 92/22646 PCl`/US92/05000
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5.1. PREPARATION OF GLOBIN GENE CONSTRUCTS
The present invention provides for a method
of producing human globin and/or hemoglobin in
transgenic pigs. Human hemoglobin is defined herein
to refer to hemoglobin~formed by globin chains encoded
human globin genes (including alpha, beta, delta,
gamma, epsilon and zeta genes) or variants thereof
lo which are naturally occurring or the products of
genetic engineering. Such variants are at least about
ninety percent homologous in amino acid sequence to a
naturally occurring human hemoglobin. In preferred
embodiments, the human hemoglobin of the invention
comprises a human alpha globin and a human beta globin
chain. The human hemoglobin of the invention
~ comprises at least two different globin chains, but
-~ may comprise more than two chains, to form, for
-~ example, a tetrameric molecule, octameric mo`ecule,
etc. In preferred embodiments of the invention, human
hemoglobin consists of two human alpha globin chains
and two human beta globin chains. As discussed infra,
the present invention also provides for hybrid
hemoglobin comprising human ~ globin and pig ~ globin.
According to particular embodiments of the
present invention, at least one human globin gene,
such as a human alpha and/or a human beta globin gene,
under the control of a suitable promoter or promoters,
is inserted into the genetic material of a pig so as
to create a transgenic pig that carries human globin
iln at least some of its red blood cells. This
requires the preparation of ap~ropriate recombinant
nucleic acid sequences. In preferred embodiments of
the invention, both human ~ and human ~ genes are
expressed. In an alternative embodiment, only human
globin is expressed. In further embodiments, human
"~
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21113~
W092/22~6 PCT/US92/05
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embryonic or fetal globin genes are expressed or are
used as developmental expression regulators of adult
genes.
Human alpha and beta globin genes may be
obtained from publicly available clones, e.g.-as
described in Swanson e~ al., 1992, Bio/Technol.
0:557-559. Nucleic acid sequences encoding human
alpha and beta globin proteins may be introduced into
an animal via two different species of recombinant
constructs, one which encodes human alpha globin, the
other encoding human beta globin; alternatively, and
preferably, both alpha and beta-encoding sequences may
be comprised in the same rec~mbinant constr~ct.
A suitable promoter, according to the
invention, is a promoter which can direct
transcription of human alpha and beta globin genes in
red blood cells. Such a promoter is preferably
selectively active in erythroid cells. This would
include, but is not limited to, a globin gene
promoter, such as the human alpha, beta, delta,
epsilon or zeta promoters, or a globin promoter from
another species. It may, for example, be useful to
¦~ utilize pig globin promoter sequences. The human
alpha and beta globin genes may be placed under the
control of different promoters, but, since it has been
inferred that vastly different levels of globin chain
production may result in lethality, it may be
preferable to place the human alpha and beta globin
genes under the control of the same promoter sequence.
In order to avoid chain imbalance and/or titration o~
transcription factors due to c~nstitutive ~-globin
promoter activity in an inappropriate cell type, it is
desirable to design a construct which leads to
3S coordinate expression of human alpha and beta globin
W092~22646 2 ~ PCT/US92/~
- 13 -
genes at the same time in development and at
quantitatively similar levels.
In one particular, non-limiting embodiment
of the invention, a construct comprising the ~
construct (also termed the "116" construct; Swanson et
al., 1992, Bio/Techno~. 10:557-559; see Figure 2A) may
be utilized. Although this construct, when present as
a transgene at high copy number, has resulted in
deleterious effects in mice, it has been used to
produce healthy transgenic pigs (see Example Section
6, infra).
In another particular, non-limiting
embodiment of the invention; a construct comprising
the ~p~ sequence (also termed the "185" construct), as
depicted in Figure lB may be used. Such a construct
has the advantage of placing both alpha and beta
,
globin-encoding sequences under the control of the
same promoter (the alpha globin promoter).
The present invention, in further specific
embodiments, provides for (i) the construct ~p~, in
which the human alpha and beta globin genes are driven
by separate copies of the human beta globin promoter
~; (Figure lC); (ii) the ~p~p~ construct, which
comprises human embryonic genes zeta and epsilon under
the control of the epsilon promoter and both alpha and
beta genes under the control of the beta promoter
(Figure lD); (iii~ the ~p~p~ construct, which
comprises human embryonic genes zeta and epsilon under
the control of the zeta promoter and both alpha and
beta genes under the control of the alpha promoter
(Figure lE); (iv) the ~p~ cons~ruct carrying a
mutation that results in an aspartic acid residue
(rather than an asparagine residue) at amino acid
number 108 of ~ globin protein, to produce hemoglobin
Yoshizuka (Figure lF); (v) the ~p~ construct carrying
:
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W092/22~ PCT/US92/05
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a mutation that results in a lysine residue (rather
than an asparagine residue) at amino acid number 108
of ~-globin protein, to produce hemoglobin
Presbyterian (Figure lG); (vi) the ~p~( ~) construct,
coinjected with LCR ~ which comprises the human ~-
globin gene under the~ontrol of the human ~-globin
promoter and a separate nucleic acid fragment
comprising the human ~-globin gene under its own
promoter (Figure lH); (vii) the ~p~ construct carrying
a mutation that results in a cysteine residue (rather
than a threonine residue) at amino acid number 134 of
~-globin protein (Figure lI); (viii) the ~p~ construct
carrying a mutation that results in a serine residue
(rather than a cysteine residue) at amino acid number
104 of the ~-globin protein, an alanine residue
(rather than a cysteine residue) at amino acid number
93 of the ~-globin protein and a valine residue
~:~ (rather than a cysteine residue) at amino acid number
~' 20 112 of the ~-globin protein ~Figure lJ); (ix) the ~p~
construct, which comprises the human adult ~-globin
promoter under its own promoter and the human ~-globin
gene under the control of the human adult ~-globin
promoter (Fig. lK); (x) Construct ~p~(~) coinjected
with LCR ~, which comprises the human ~-globin gene
: under the control of the human ~-globin promoter and a
separate nucleic acid fragment comprising the human ~-
globin gene under its own promoter (Fig. lL~; (xi)
Construct LCR ~ coinjected with LCR ~, which
comprises the human ~-globin gene under the control of
, ~ its own promoter and a separate nucleic acid fragment
comprising the human embryonic~~-globin gene and the
adult ~-globin gene under the control of their own
promoters (Fig. lM); (xii) the ~p~ construct carrying
a mutation that results in a methionine residue
(rather than a lysine residue) at amino acid number 61
,~
-~W092/22~6 ~ 4 3 PCT/~S92/~N~
-- 15 --
of the a-globin protein (Fig. lN); (xiii) the ~
construct, which comprises the human embryonic epsilon
gene, the human adult alpha globin gene and the human
adult beta globin gene linked in tandem from 5'- to 3'
(Fig. lO); (xiv) the ~ construct, which comprises
the human adult alp~a-~lobin gene, the human embryonic
epsilon globin gene and the human adult beta globin
gene linked in tandem from 5'- to 3' (Fig. lP); (xv)
lo the ~ construct, which comprises two copies of the
human adult alpha-globin gene, the human embryonic
epsilon globin gene and the human adult beta globin
gene linked in tandem from 5'- to 3' (Fig. lQ); (xvi)
the ~ p)~ construct, which comprises the human
adult alpha-globin gene, the human embryonic epsilon
~ globin gene and the human adult beta globin gene under
; the control of the endogenous porcine adult beta-` globin promoter all linked in tandem from 5'- to 3'
`~ (Fig. lR); (xvii) the ~p~ construct carrying a
' 20 mutation that results in a cysteine residue (rather
than an aspartic acid residue) at amino acid number 75
of the a-globin protein (Fig. lS); (xviii) the ap~
construct carrying a mutation that results in an
arginine residue (rather than a tyrosine residue) at
amino acid number 42 at the ~-globin protein (Fig.
lT); (xvix) the LCR ~a construct, which comprises
the human embryonic epsilon globin gene, the human
adult beta globin gene and two copies of the human
adult alpha-globin gene linked in tandem from 5'- to
3' (Fig. lU); (xx) the LCR ~ construct, which
comprises the human embryonic epsilon globin gene, the
human adult beta globin gene a~d the human adult
alpha-globin gene linked in tandem from 5'- to 3'
(Fig. lV); (xxi) the ap~ construct carrving a mutation
that results in a lysine residue (rather than a
tyrosine residue) at amino acid number 42 of the a
~, ~
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wo gt,2~1 1 3 4 8 PCT/US92/~
- 16 -
globin protein (Fig. lW); (xxii) the ap~ construct
carrying a mutation that results in an arginine
residue (rather than a tyrosine residue) at amino acid
number 42 at the ~-globin protein and a glutamic acid
residue (rather than an aspartic acid residue) at
amino acid number 99 o~ the ~-globin protein (Fig.
lX); and (xxiii) the ~p~ construct carrying a mutation
that results in a lysine residue (rather than a
tyrosine residue) at amino acid number 42 of the ~-
globin protein and a glutamic acid residue (rather
than an aspartic acid residue) at amino acid number 99
of the ~-globin protein (~ig. lY).
The recombinant nucleic acid constructs
described above may be inserted into any suitable
plasmid, bacteriophage, or viral vector for
amplification, and may thereby be propagated using
~ methods known in the art, such as those described in
`~ Maniatis et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor, N.Y. In the working
examples presented below, the PUC vector (Yanish-
-~ Perron et al., 1985, Gene 103-119) was utilized.
Constructs may desirably be linearized for
preparation of transgenic pigs. Vector sequence may
desirably be removed.
5. 2 . P~PARATION OF TRANSGENIC PIGS
The recombinant constructs described above
may be used to produce a transgenic pig by any method
known in the art, including but not limited to,
microinjection, embryonic stem (ES) cell manipulation,
electroporation, cell gun, transfection, transduction,
retroviral infection, etc. Species of constructs may
be introduced individually or in groups of two or more
types of construct.
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According to a preferred specific embodiment
of the invention, a transgenic pig may be produced by
the methods as set forth in Example Section 6, infra.
Briefly, estrus may be synchronized in sexually mature
gilts (>7 months of age) by feeding an orally active
progestogen (allyl tre*bolone, AT: lS mg/gilt/day) for
12 to 14 days. On the last day of AT feeding all
gilts may be given an intramuscular injection (IM) of
$0 prostaglandin F~ (Lutalyse: 10 mg/injection) at 0800
and 1600 hours. Twenty-four hours after the last day
of AT consumption all donor gilts may be administered
a single IM injection of pregnant mare serum
gonadotropin (PMSG: 1500 IU).~ Human chorionic
gonadotropin (HCG: 7S0 IU) may be administered to all
donors at 80 hours after PMSG.
Following AT withdrawal, donor and recipient
~ gilts may be checked twice daily for signs of estrus
-~ using a mature boar. Donors which exhibited estrus
within 36 hours following HCG administration may be
~- bred at 12 and 24 hours after the onset of estrus
using artificial and natural (respectively)
insemination.
` Between 59 and 66 hours after the
-~ 25 administration of HCG one- and two-cell ova may be
surgically recovered from bred donors using the
following procedure. General anesthesia may be
induced by administering 0.5 mg of acepromazine/kg of
bodyweight and 1.3 mg ketamine/kg of bodyweight via a
peripheral ear vein. Following anesthetization, the
` reproductive tract may be exteriorized following a
mid-ventral-laparotomy. A dràwn glass cannula (O.D. 5
mm, length 8 cm) may be inserted into the ostium of
the oviduct and anchored to the infundibulum using a
single silk t2-0) suture. Ova may be flushed in
~1 retrograde fashion by inserting a 20 g needle into the
SlJBs~T~
W092/22~ 2 11 1~ PCT/US92/~
- 18 -
lumen of the oviduct 2 cm anterior to the uterotubal
junction. Sterile Dulbecco's phosphate buffered
saline (PBS) supplemented with 0.4% bovine serum
albumin (BSA) may be infused into the oviduct and
flushed toward the qlass cannula. The medium may be
collected into ster~le l7 x 100 mm polystyrene tubes.
Flushings may be transferred to lO x 60 mm petri
dishes and searched at lower power (50 x) using a Wild
N3 stereomicroscope. All one- and two-cell ova may be
washed twice in Brinster's Modified Ova Culture-3
medium (BMOC-3) supplemented with 1.5% BSA and
transferred to 50 ~1 drops of BMOC-3 medium under oil.
Ova may be stored at 38OC under a 90% N2, 5% o~, 5% Co2
atmosphere until microinjection is performed.
one- and two-cell ova may be placed in a
Eppendorf tube (15 ova per tube) containing 1 ml HEPES
Medium supplemented with 1.5% BSA and centrifuged for
~ 6 minutes at 14000 x g in order to vi~ualize pronuclei
`~ 20 in one-cell and nuclei in two-cell ova. Ova may then
be transferred to a 5 - 10 ~1 drop of HEPES medium
~ under oil on a depression slide. Microinjection may
; be performed using a Laborlux microscope with
Nomarski optics and two Leitz micromanipulators. 10-
1700 copies of construc~ DNA (linearized at a
concentration of about lng/~l of Tris-EDTA buffer) may
be injected into one pronuclei in one-cell ova or both
nuclei in two-cell ova.
Microinjected ova may be returned to
microdrops of BMOC-3 medium under oil and maintained
at 38C under a 90% N2, 5% CO~, 5% 2 atmosphere prior
to their transfer to suitable-`recipients. Ova may
preferably be transferred within 10 hours of recovery.
only recipients which exhibit estrus on the
same day or 24 hours later than the donors may
preferably be utilized for embryo transfer.
,
8UBSTrrU
-~W092r22~ 2 1 ~ ~ 3 ~ 8 PCT/US92~0~
-- 19 --
Recipients may be anesthetized as described earlier.
Following exteriorization of one oviduct, at least 30
injected one-and/or two-cell ova and 4-6 control ova
may be transferred in the following manner. The
tubing from a 21 g x 3/4 butterfly infusion set may be
connected to a 1 cc syringe. The ova and one to two
mls of BMOC-3 medium may be aspirated into the tubing.
The tubing may then be fed through the ostium of the
oviduct until the tip reaches the lower third or
isthmus of the oviduct. The ova may be subsequently
expelled as the tubing is slowly withdrawn.
The exposed portion of the reproductive
tract may be bathed in a stèrile 10% glycerol-0.9%
saline solution and returned to the body cavity. The
connective tissue encompassing the linea alba, the fat
and the skin may be sutured as three separate layers.
- An uninterrupted Halstead stitch may be used to close
the lina alba. The fat and skin may be closed using a
~ 20 simple continuous and mattress stitch, respectively.
-~ A topical antibacterial agent (e.g. Furazolidone) may
then be administered to the incision area.
Recipients may be penned in groups of about
four and fed 1.8 kg of a standard 16% crude protein
corn-soybean pelleted ration. Beginning on day 18
(day 0 = onset of estrus), all recipients may be
checked daily for signs of estrus using a mature boar.
on day 35, pregnancy detection may be performed using
ultrasound. On day 107 of gestation recipients may be
transferred to the farrowing suite. In order to
ensure attendance at farrowing time, farrowing may be
induced by the administration-~f prostaglandin F2a t10
mg/injection) at 0800 and 1400 hours on day 112 of
gestation. In all cases, recipients mav be expected
to farrow within 34 hours following PGF2a
administration.
:~ . S(IB23~
wo 92,22~6 2 1 1 1~ ~ PCT~US92/05~
- 20 -
Twenty-four hours after birth, all piglets
may be processed, i.e. ears notched, needle teeth
clipped, 1 cc of iron dextran administered, etc. A
tail biopsy and blood may also be obtained from each
pig.
Pigs produce~ according to this method are
described in Example Section 6, infra, and are
depicted in Figure 2. Such pigs are healthy, do not
appear to be anemic, and appear to grow at a rate
comparable to that of their non-transgenic
littermates. Such pigs may transmit the transgene to
their offspring.
Pigs having certain characteristics may be
especially useful for the production of human
hemoglobin; such pigs, examples of which follow,
represent preferred, non-limiting, specific
;~ embodiments of the invention.
According to one preferred specific
embodiment of the invention, a transgenic pig contains
at least twenty copies of a glo~in transgene.
According to a second preferred specific
~ embodiment, the P50 of whole blood of a transgenic pig
; according to the inv~ntion is increased by at least
ten percent over the P50 of the whole blood of a
comparable non-transqenic pig,taking into
consideration factors such as altitude, oxygen
concentrations, pregnancy, the presence of mutant
hemoglo~in, etc. Thus, the presen~ invention provides
for a non-pregnant transgenic pig that carries and
expresses a human globin transgene in which the P~ of
whole blood of the transgenic`~ig is at least ten
percent greater than the P~" of whole blood of a
comparable non-pregnant non-transgenic pig at the same
altitude.
:
:':
SUBsrlT
~W092/22~ 2 1 ~ ~ 3 ~ ~ PCT/US92/05~
- 21 -
In other preferred specific embodiments, the
present invention provides for a transgenic pig in
which the amount of human globin produced relative to
total hemoglobin is at least two percent, more
preferably at least five percent, and most preferably
at least ten percent.~-
Section 6, infra, describes transgenic pigswhich serve as working examples of preferred, non-
~0 limiting, specific examples of the invention.
.3. PREPARATION OF HUMAN HEMOGLOBIN AND
ITS SEPARATION FROM PIG HEMOGLOBIN
The present invention provides fQr a method
for producing human hemoglobin comprising introducing
a transgene or transgenes encoding human hemoglobin,
such as a human alpha globin and a human beta globin
gene, under the control of a ~uitable promoter or
;
promoters, into the genetic material of a pig so as to
create a transgenic pig that expresses human
hemoglobin in at least some of its blood cells.
The present invention also provides for a
method of producing human hemoglobin comprising (i)
introducing a human alpha globin and a human beta
globin gene, under the control of a suitable promoter
or promoters, into the genetic material of a pig so as
to create a transgenic pig that expresses human
hemoglobin in at least some of its red blood cells;
(ii) collecting red blood cells from the transgenic
pig; (iii) releasing the contents of the collected red
blood cells to form a lysate; (iv) subjecting the
lysate of the red blood cells~o a purification
procedure that substantially separates human
hemoglobin from pig hemoglobin; and (v) collecting the
fractions that contain purified human hemoglobin.
Such fractions may be identified by isoelectric
focusing in parallel with appropriate standards. In a
,~
S(JBsT
W092/22~ 2 ~ 22 - PCT/US9~/05
preferred embodiment of the invention, human
hemoglobin may be separated from pig hemoglobin by
DEAE anion exchange column chromatography.
In order to prepare human hemoglobin from
the transgenic pigs described above, red blood cells
are obtained from the~pig using any method known in
the art. The red blood cells are then lysed using any
method, including hemolysis in a hypotonic solution
such as distilled water, or using techniques as
described in 1981, Methods in Enzymology Vol. 76,
and/or tangential flow filtration.
For purposes of ascertaining whether human
hemoglobin is being produced~by a particular
transgenic pig, it may be useful to perform a small-
scale electrophoretic analysis of the hemolysate, such
as, for example, isoelectric focusing using standard
~ techniques.
r~ Alternatively, or for larger scale
.,, ~
purification, human hemoglobin may be separated from
pig hemoglobin using ion exchange chromatography.
Surprisingly, as discussed in Section 7, su~ra, human
hemoglobin was observed to readily separate from pig
~ hemoglobin using ion exchange chromatography whereas
-~ 25 mouse hemoglobin and human hemoglobin were not
- separable by such methods. Any ion exchange resin
known in the art or to be developed may be utilized,
including, but not limited to, resins comprising
diethylaminoethyl, Q-Sepharose, QCPI (I.B.F.) Zephyr,
3Q Spherodex, ectiola, carboxymethylcellulose, etc.
provided that the resin results in a separation of
human and pig hemoglobin comparable to that achieved
` using DEAE resin.
According to a specific, nonlimiting
3S embodiment of the invention, in order to separate
human from pig hemoglobin (including humantpig
~,
3UBSTITUTE S~F~T
s W092t22~ 2 1 :1 1 3 1 ~ PCT/US92/~
- 23 -
hemoglobin hybrids) to produce substantially pure
human hemoglobin, a hemolysate of transgenic pig red
blood cells, prepared as above may be applied to a
DEAE anion exchange column equilibrated with 0.2 M
glycine buffer at pH 7.~ and washed with 0.2 M glycine
p~ 7.8/5 mM NaCl, and~ay then be eluted with a 5-30
mM NaCl gradient, or its equivalent (see, for example,
Section 9 infra). Surprisingly, despite about 85
percent homology between human and pig globin chains,
` human and pig hemoglobin separates readily upon such
treatment, with human hemoglobin eluting earlier than
pig hemoglobin. Elution may be monitored by optical
density at 405 nm and/or ele~trophoresis of aliquots
taken from serial fractions. Pig hemoglobin, as well
as tetrameric hemoglobin composed of heterodimers
; formed between pig and human globin chains, may be
s-parated from human hemoglobin by this method. Human
hemoglobin produced in a transgenic pig and separated
~-~ 20 from pig hemoglobin by this method has an oxygen
binding capability similar to that of native human
hemoglobin.
According to another specific, non-limiting
; ~
embodiment of the invention, human hemoglobin may be
separated from pig hemoglobin (including human/pig
hemoglobin hybrids) using QCPI ion exchange resin as
follows:
About 10 mg of hemoglobin prepared from
transgenic pig erythrocytes may be diluted in 20ml of
Buffer A (Buffer A = lOmM Tris, 20mM Glycine pH 7.5).
This 2Oml sample may then be loaded at a flow rate of
about Sml/min onto a QCPI column (10 ml) which has
been equilibrated with Buffer A. The column may then
~ ~ be washed with 2 volumes of Buffer A, and then with 20
`~ 35 column volumes of a 0-50mM NaCl gradient (10 column
j~ volumes of Buffer A ~ 10 column volumes of lOmM Tris,
~ "
.,..,.~.,
~,,
,~
"~
SUB5 rl'r ~
Wo 92/22646 ?~ , 3 ~ Cr/US92/05000 ~ -;
-- 24 --
20mM Glycine, 50mM NaCl Ph 7.5) or, alteratively, 6
column volumes of 10mM Tris, 20mM Glycine, 15mM NaCl,
pH 7.5, and the O.D.28(, absorbing material may be
collected in fractions to yield the separated
hemoglobin, human hemoglobin being identified, for '
example, by isoelectr~c focusing using appropriate
standards. The QCPI column may be cleaned by elution
with 2 column volumes of 10mM Tris, 20mM Glycine, lM
-~10 NaCl, pH 7.s.
5.4. PREPARATION OF HUMAN/PIG HYBRID HEMOGLOBIN
~The present invention also provides for
- ~essentially purified and isolated human/pig hybrid
hemoglobin, in particular human ~/pig ~ hybrid
hemoglobin. Pig ~/human ~ hybrid has not been
observed to form either n vitro in reassociation
experiments or n vitro in transgenic pigs.
The present invention provides for hybrid
~20 hemoglobin and its use as a blood substitute, and for
s~a pharmaceutical composition comprising the
essentially purified and isolated'human/pig hemoglobin
hybrid in a suitable pharmacological carrier,
~Hybrid hemoglobin may be prepared from
-~ ; 25 transgenic pigs, as described herein, and then
purified by chromatography, immunoprecipitation, or
any other method known to the skilled artisan. The
use of isoelectric focusing to separate out hemoglobin
hybrid is shown in Figures 3 and 5.
Alternatively, hybrid hemoglobin may be
prepared using nucleic acid constructs that comprise
; both human and pig globin sequences which may then be
'~ expressed in any suitable microorganism, cell, or
~ transgenic animal. For example, a nucleic acid
'~3S construct that comprises the human ~ and pig ~ globin
~genes under the control of a suitable promoter may be
., ~
~^~, ~,
"~
SUBsrm~ r~_
~ ,W092/22~ 2 1 1 1 3 ~ ~ pCT/US92/05~
- 25 -
expressed to result in hybrid hemoglobin. As a
specific example, human ~ globin and pig ~ globin
genes, under the control of cytomegalovirus promoter,
may be transfected into a mammalian cell such as a COS
cell, and hybrid hemoglobin may be harvested from,such
cells. Alternativety,'~~such constructs may be ,
expressed in yeast or bacteria.
It may be desirable to modify the hemoglobin
hybrid so as to render it non-immunogenic, for
example, by linkage with polyethylene glycol or by
encapsulating the hemoglobin in a membrane, e.g. in a
liposome.
'
6. EXAMPLE: GENERATION OF TRANSGENIC PIGS
THAT PRODUCE HUMAN HEMOGLOBIN
6.1. MATERIAL$ AND METHODS
6.1.1. NUCLEIC ACID CONSTRUCTS
~- Constructs 116 (the ~~ construct), 185 (the
~p~ construct), or 263 ~the ~p~ construct) were
microinjected into pig ova as set forth below in order
to produce transgenic pigs.
6.1.2. PRODUCTION OF TRANSGENIC PIGS
' Estrus was synchronized in sexually mature
gilts (>7 months of age) by feeding an orally active
progestogen (allyl trenbolone, AT: 15 mgtgilt/day) for
12 to 14 days. On the last day of AT feeding all
gilts received an intramuscular injection (IM) of
prostaglandin F~ (Lutalyse: 10 mg/in~ection) at 0800
and 1600. Twenty-four hours after the last day of AT
consumption all donor gilts re~,eived a single IM
injection of pregnant mare ~erum gonadotropin (PMSG:
1500 IU). Human chorionic gonadotropin (HCG: 750 IU)
was administered to all donors at 80 hours after PMSG.
Following AT withdrawal, donor and recipient
gilts were checked twice daily for signs of estrus
:
.
8UBS~TI-r ~
W092/22~ 2 1 ~ PCT/US92tO5~0 -
- 26 -
using a mature boar. Donors which exhibited estrus
within 36 hours following HCG administration were bred
at 12 and 24 hours after the onset of estrus using
artificial and natural (respectively) insemination.
Between 59 and 66 hours after the
administration of HCG~ one- and two-cell ova were
surgically recovered from bred donors using the
following procedure. General anesthesia was induced
by administering 0.5 mg of acepromazine/kg of
bodyweight and 1.3 mg ketamine/kg of bodyweight via a
peripheral ear vein. Following anesthetization, the
reproductive tract was exteriorized following a mid-
ventral laparotomy. A drawn~glass cannula (O.~. 5 mm,
length 8 cm) was inserted into the ostium of the
oviduct and anchored to the infundibulum using a
single silk (2-0) suture. Ova were flushed in
retrograde fashion by inserting a 20 g needle into the
lumen of the o~iduct 2 cm anterior to the uterotubal
junction. Sterile Dulbecco's phosphate buffered
saline (PBS) supplemented with 0.4% bovine serum
albumin (BSA) was infused into the oviduct and flushed
toward the glass cannula. The medium was collected
into sterile 17 x 100 mm polystyrene tubes. Flushings
were transferred to 10 x 60 mm petri dishes and
searched at lower power (50 x) using a Wild M3
stereomicroscope. All one- and two-cell ova were
washed twice in Brinster's Modified Ova Culture-3
medium ~BMOC-3) supplemented with 1.5% BSA and
transferred to 50 ~l drops of BMOC-3 medium under oil.
Ova were stored at 38~C under a 90~ N2, 5~ 0~, 5% CO,
atmosphere until microinjection was performed.
One- and two-cell ova were placed in an
Eppendorf tube (15 ova per tube) containing 1 ml HEPES
Medium supplemented with 1.5% BSA and centrifuged for
6 minutes at 14000 x g in order to visualize pronuclei
SlJB~I~ r~ ~
.~W092/22~ 2 1 1 1 ~ ~ 8 PCT/US92/~
~" ?
-- 27 --
in one-cell and nuclei in two-cell ova. Ova were then
transferred to a 5 -10 ~l drop of HEPES medium under
oil on a depression slide. Microinjection was
performed using a Laborlux microscope with Nomarski
optics and two Leitz micromanipulators. 10-1700
copies of construct DN~ (lng/~l of Tris-EDTA buffer)
were injected into one pronuclei in one-cell ova or
- ~ both nuclei in two-cell ova.
` Microinjected ova were returned to
microdrops of BMOC-3 medium under oil and maintained
at 38C under a 90% N~, 5% C02, 5% O, atmosphere prior
to their transfer to suitable recipients. Ova were
transferred within 10 hours ~f recovery.
Only recipients which exhibited estrus on
the same day or 24 hours later than the donors were
utilized for embryo trdnsfer. Recipients were
anesthetized as described earlier. Following
exteriorization of one oviduct, at least 30 injected one-
and/or two-cell ova and 4-6 control ova were
transferred in the following manner. The tubing from
a 21 g x 3/4 butterfly infusion set was connected to a ;
~ .
1 cc syringe. The ova and one to two mls of BNOC-3
medium were aspirated into the tubing. The tubing was
then fed through the ostium of the oviduct until the
tip reached the lower third or isthmus of the oviduct.
The o~a were subsequently expelled as the tubing was
slowly withdrawn.
The exposed portion of the reproductive
tract was bathed in a sterile 10% glycerol-0.9% saline
~solution and returned to the body cavity. The
connective tissue encompassinq~the linea alba, the fat
and the skin were sutured as three separate layers.
An uninterrupted Halstead .stitch was used to close the
lina alba. The fat and skin were closed using a
simple continuous and mattress stitch, respectively.
~ '
8~1B~
21i1~
W092/22~ PCT/VS92~05~0
- 28 -
A topical antibacterial agent (Furazolidone) was then
administered to the incision area. -
Recipients were penned in groups of four and
fed 1.8 kg of a standard 16% crude protein corn-
soybean pelleted ration. Beginning on day 18 (day 0
onset of estrus), all~recipients were checked daily
for signs of estrus using a mature boar. On day 35,
pregnancy detection was performed using ultrasound.
~0 On day 107 of gestation recipients were transferred to ~-
the farrowing suite. In order to ensure attendance at
farrowing time, farrowing was induced by the
administration of prostaglandin F2, (10 mg/injection)
at 0800 and 1400 hours on da~ 112 of gestation. In `~
all cases, recipients farrowed within 34 hours
following PGF2a administration.
Twenty-four hours after birth, all piglets
were processed, i.e. ears were notched, needle teeth
clipped, 1 cc of iron dextran was administered, etc.
A tail biopsy and blood were also obtained from each
pig- -
6.2. RESULTS AND DISCUSSION
Of 3566 injected ova, thirteen transgenic
pigs that expressed human hemoglobin were born, two ofwhich died shortly after birth due to normal breeding-
related incidents completely unrelated to the fact
that they were transgenic pigs ~Table I). The
remaining 11 have appeared to be healthy. A
photograph of one transgenic pig is presented in
Figure 2. Profiles of the pigs and of the percent
"authentic" and "hybrid" human hemoglobin ("HB")
produced are set forth in Table II, infra. Total
hemoglobin was calculated as the sum of human ~ plus
one-half of the human ~ pig ~ hybrid. Figure 3
presents the results of isoelectric focusing and
~ W092~22~6 2 1 L 1 3 ~ ~ PCT/US92/05~0
- 29 -
tritcn acid urea gels of hemoglobin produced by three
of these pigs (numbers 12-1, 9-3, and 6-3) which
demonstrate the expression of human alpha and beta
globin in these animals.
i ~ TA~LE I
Efficiency of Transgenic Pig Production
Human Hemoglobin Gene Construct(s)
Total After
Parameter 22 Trials :.
Total Ova Collected 8276
Total # Fertilized 7156
lS Total # Injected 3566
# Injected Ova Transferred 3566
# Control Ova Transferred 279
# Recipients Used 104
# Pigs Born (Male, Female) 208,332
# Transgenic (Male, Female) 8,5 (0.36)a
# Expressing 13 --
a Proportion of injected ova which developed into
transgenic pigs (13 transgenics/3566 injected ova).
~`
SlJBsT1Tl JT~ ~L~
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WO 92/22646 PCI`/US92/05000 ~ `
- 3 0 -
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Q. .~
-~;40 g2/22~6 2 1 1 ~ 3 ~ ~ PCT/US92/05~
- 31 -
Table III presents the profiles of offspring
of pig number 9-3, which shows that the F1 generation
of transgenic pigs are capable of expressing
hemoglobin. Of note, none of the offspring of pig
number 6-3 were found to be transgenic, possibly due
to the absence of transgene in the animal's
reproductive tissue.
",
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WO 92/22646 - 3 2 - PCr/US92/0501~0~
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;-~ W092/22~6 PCT/US92/OS~0
- 33 -
The birth weights of the transgenic pigs
have been approximately equivalent to the birth
weights of their non-transgenic littermates. As the
transgenic pigs matured, their weights remained
comparable to the weights of control animals.
7. EXAMPLE: SEPARATION OF HUMAN HEMOGLOBIN
FROM PIG HEMOGLOBIN 8Y DEAE CHROMATOGRAPHY
7.1. MATERIALS AND METHODS
7.1.1. PURIFICATION BY DEAE CHROMATOGRAPHY
For purification, red blood cells were
collected by centrifugation of 5000 rpm for 3 minutes -~
in an eppendorf microcentrifuge and washed three times
~5 with an equal volume (original blood) of 0.9% NaCl.
Red cells were lysed with 1.5 volumes deionized H2O,
centrifuged at 15,000 rpm, and the supernatant was
fractionated by anion exchange chromatography. DEAE
cellulose chromatography (DE-SE manufactured by
2~ Whatman, Ltd.) was performed according to W. A.
Schroeder and T. H. J. Huisman "The Chromatography of
Hemoglobin", Dekker, New York, pp. 74-77. The 0.25 ml
red cell hemolysate described above wa~ applied to 1
cm x 7 cm DE-52 column pre-equilibral:ed in 0.2 M
glycine pH 7.8 and was washed with 5 column volumes of
0.2 M glycine pH 7.8/5 mM NaClO HPmoglobin were
eluted with a 200 ml S-30 mM NaCl/0.2 M glycine pH 7.8
gradient. To complete elution of pig hemoglobin, an
additional 50 to 100 ml of 30 mM CaCl/glycine pH 7.8
was added to the column. Elution of hemoglobin was
monitored by absorbance of 415 mM and by IEF analysis
of column fractions.
7.1.2. REASSOCIATION OF GLOBIN CHAINS
Reassociation of globin chains was performed
essentially as described in Methods in Enzymol. `
3 ~ ~ :W092./22~6 PCT/US92/O~W~
- 34 -
76:126-133. 25 lambda of pig blood, 25 lambda of
human blood,or a 25 lambda mixture of 12.5 lambda
human blood and 12.5 lambda pig blood were treated as
follows. The blood was pelleted at a setting of 5 on
microfuge for 2 minutes, then washed three times with
100 lambda 0.9 percen~-~NaCl. The cells were lysed
with 50 lambda H20, then spun at high speed to confirm
lysis. 50 lambda of the lysed cells was then combined
with 50 lambda 0.2 M Na Acetate, pH 4.5, put on ice
and then incubated in a cold room overnight. After
adding 1.9 ml 0.1 M NaH2PO44, pH 7.4 each sample was
spun in centricon tubes at 4C and 5K until about 0.5
ml remained. Then 1 ml of 0.~1 M NaH~PO4 pH 7.4 was
added and spun through at about SK until about 0.2 ml
volume was left. The hemoglobin was then washed from
the walls of the centricon tube, an eppendorf adaptor
was attached, and a table top microfuge was used to
remove each sample from its centricon tube. The
samples were then analyzed by isoelectric focusing.
7.2. RESULTS AND DISCUSSION
7.2.1. HUMAN AND PIG HEMOGLO~IN WERE SEPARATED
FROM A HEMOLYZED MIXTURE OF HUMAN AND
PIG BLOOD
Equal proportions of human and of pig blood
were mixed and lysed, and the resulting hemolysate was
subjected to DEAE chromatography as described supra.
As shown in Figure 4A, pig hemoglobin separated
virtually completely from human hemoglobin. This
complete separation is surprising in light of the
structural similarity between human and pig
hemoglobin; pig and human alpha globin chains are 84.4
percent homologous and pig and human beta globin
chains are 84.9 percent homologous. It is further
surprising because, as shown in Figure 4C, when human
and mouse blood was mixed, hemolyzed, applied to and
SLJB~3 . n . .~
~ W092/22~ 21 113 4 ~ PCT/USg2~0~
- 35 -
eluted from a DEAE column according to methods set
forth in Section 7.1.1., supra, human and mouse
hemoglobin were not observed to separate despite the
fact that mouse and human alpha globin chains are
about 85.8 percent homologous and mouse and human beta
globin chains are 80.~-~percent homologous. The ease of
separation of human and pig hemoglobin on DEAE resin
appears to be both efficient and economical.
Interestingly, the order of elution of the
proteins from the anion exchange column was not as
expected. Based on the relative pI's of the proteins
as deduced from the IEF gels, the predicted order of
elution would be first the hybrid (human a/pig ~)
15 followed by the authentic human ~/human ~. The last ~-
protein to elute from the anion exchange column then
would be the endogenous pig ~/pig ~ protein. However,
under all the conditions currently attempted the order
of elution was altered such that the human hemoglobin
was the first to elute. The second peak was an
enriched fraction of the hybrid followed very closely
by the pig hemoglobin.
7.2.2. HUMAN AND PIG HEMOGLOBIN AND HUMAN/PIG
HETEROLOGOUS HEMOGLOBIN WERE SEPARATED
FROM HEMOLYSATE PREPARED FROM A
TRANSGENIC PIG _ _
Blood from transgenic pig 6-3 (as described
in Section 6, supra) was lysed by hypotonic swelling
and the resulting hemolysate was subjected to DEAE
chromatography as described su~ra. As shown in Figure
4B, human hemoglobin was separated from pig hemoglobin
and from human ~ globin/pig bàta globin heterologous
hemoglobin. As shown in Figure 4D, human hemoglobin
was substantially purified by this method.
S~JB~
W092/22~6 2 1 1 1 ~ ~ 8 PCT/US92/05~
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7.2.3. PIG ALPHA GLOBIN/HUMAN BETA GLOBIN
HETEROLOGOUS HEMOGLOBIN DOES NOT
APPEAR TO FORM BASED ON REASSOCIATION
DATA
Heterologous association between pig alpha
globin and human beta~lobin chains has not been
detected in hemolysates obtained from human
hemoglobin-expressing transgenic pigs. It was
possible, however, that this observation could be
explained by relatively low levels of human beta
globin expression. Alternatively, association between
pig alpha globin and human beta globin may be
chemically unfavorable. In order to explore this
possibility, reassociation experiments were performed
in which pig and human hemoglobin were mixed,
dissociated, and then the globin chains were allowed
to reassociate. As shown in the isoelectric focusing
gels depicted in Figure 5, although pig ~/pig ~, human
~/human ~, and human ~pig ~ association was observed,
no association between pig ~ globin and human ~ globin
appeared to have occurred. Therefore the pig ~/human
heterologous hemoglobin should not be expected to
complicate the purification of human hemoglobin from
transgenlc pigs.
8. EXAMPLE: SEPARATION OF HUMAN
HEMOGLOBIN FROM PIG HEMOGLOBIN
BY OCPI CHRO~TOGRAPHY
8.1. MATERIALS AND METHODS
30 `r Clarified hemolysate from transgenic pig 6-3
13mg/ml; Buffer A: 10mM Tris, 20mM Glycine pH 7.5;
Buffer B: 10mM Tris, 20mM Glycine, 15 mM NaCl pH 7.5;
Buffer C: 10mM Tris, 20mM Glycine, lM NaCl pH 7.5;
Buffer D: 10mM Tris, 20mM Glycine, 50 mM NaCl pH 7.5;
QCPI column 10ml Equili~rated in Buffer A; Trio
purification system. 10mg Ot hemoglobin prepared from
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-~W092/22~6 2 1 ~ 1 3 4 ~ PCT/US92/05~ ;
_ 37 _
transgenic pig 6-3 was diluted in 20ml Buffer A. 20ml
of sample was loaded at a flow rate of 5ml/min onto
the QCPI column, and washed with 2 column volumes of
Buffer A. The column was then washed with 20 column
volumes of a 0-50mM NaCl gradient. (10 column volumes
Buffer A + 10 column volumes of Buffer D) and the
O.D.280 absorbing material was collected. The column
was then cleaned with 2 column volumes of Buffer C,
and then re-equilibrated with 2 column volumes of
Buffer A. -
8.2. RESULTS
Analysis of the W trace (peak vs. volume of
gradient) (Fig. 6) revealed that the human hemoglobin
was eluted at 15 mM NaCl. Subsequent purifications
have been performed utilizing the same protocol as `
~ above, only using 6 column volumes of Buffer B (15m~
- NaCl~ to elute the human hemoglobin rather than the
gradient. In addition, non-transgenic pig
chromatographed by this method does not elute from the
QCPI with Buffer B, while native human hemoglobin
does. The protein that eluted at 15mM NaCl was
analyzed on the Resolve isoelectric focussing system
and found to be essentially pure of contaminating pig
hemoglobin or hybrid hemoglobin.
9. EXANPLE: HUMAN ALPHA/PIG BETA GLOBIN
HYBRID HEMOGLOBIN EXHIBIT INCREASED P5(,
As shown in Tables II and III, supra,
transgenic pigs of the invention were all found to
produce significant amounts o~human ~/pig ~ globin
hybrid hemoglobin (the pig ~/human ~ hybrid was not
observed). Significantly, pigs that expressed higher
percentages of hybrid also appeared to exhibit `
elevated P50 values for their whole blood (Figure 7).
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Various publications are cited herein which
are hereby incorporated by reference in their
entirety.
s
~:
S~lB~
