U.S. patent application number 10/463980 was filed with the patent office on 2004-01-29 for exogenous proteins expressed in avians and their eggs.
Invention is credited to Harvey, Alex J., Ivarie, Robert D., Liu, Guodong, Morris, Julie A., Rapp, Jeffrey C..
Application Number | 20040019923 10/463980 |
Document ID | / |
Family ID | 32829426 |
Filed Date | 2004-01-29 |
United States Patent
Application |
20040019923 |
Kind Code |
A1 |
Ivarie, Robert D. ; et
al. |
January 29, 2004 |
Exogenous proteins expressed in avians and their eggs
Abstract
This invention provides vectors and methods for the stable
introduction of exogenous nucleic acid sequences into the genome of
avians in order to express the exogenous sequences to alter the
phenotype of the avians or to produce desired proteins. In
particular, transgenic avians are produced which express exogenous
sequences in their oviducts and which deposit exogenous proteins
into their eggs. Avian eggs that contain exogenous proteins are
encompassed by this invention. The instant invention further
provides novel forms of interferon and erythropoietin which are
efficiently expressed in the oviduct of transgenic avians and
deposited into avian eggs.
Inventors: |
Ivarie, Robert D.;
(Watkinsville, GA) ; Harvey, Alex J.; (Athens,
GA) ; Morris, Julie A.; (Watkinsville, GA) ;
Liu, Guodong; (Mississagua, CA) ; Rapp, Jeffrey
C.; (Athens, GA) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER
EIGHTH FLOOR
SAN FRANCISCO
CA
94111-3834
US
|
Family ID: |
32829426 |
Appl. No.: |
10/463980 |
Filed: |
June 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10463980 |
Jun 17, 2003 |
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10351196 |
Jan 24, 2003 |
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10351196 |
Jan 24, 2003 |
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09173864 |
Oct 16, 1998 |
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60062172 |
Oct 16, 1997 |
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Current U.S.
Class: |
800/19 ; 119/300;
435/456 |
Current CPC
Class: |
C12N 2830/90 20130101;
C12N 2840/20 20130101; C12N 2740/13043 20130101; C12N 2830/15
20130101; C12N 15/902 20130101; C12N 2830/40 20130101; C12N
2840/203 20130101; A01K 2267/01 20130101; C12N 2799/027 20130101;
C12N 15/8509 20130101; A01K 2227/30 20130101; C12N 15/86 20130101;
A01K 2217/05 20130101; C12N 2830/008 20130101 |
Class at
Publication: |
800/19 ; 435/456;
119/300 |
International
Class: |
A01K 067/027; C12N
015/867 |
Claims
What is claimed is:
1. A vector comprising a coding sequence and a promoter in
operational and positional relationship to express said coding
sequence in an avian oviduct, wherein said coding sequence is
selected from the group consisting of recombinant transgenic
poultry derived interferon-.alpha. 2b and erythropoietin coding
sequence.
2. The vector of claim 1, wherein said promoter is selected from
the group consisting of a cytomegalovirus (CMV) promoter, a MDOT
promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia
virus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter,
an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter,
an ovomucoid promoter, an ovomucin promoter, an ovotransferrin
promoter, and segments thereof, wherein said promoter is sufficient
for effecting expression of the coding sequence in the avian
oviduct.
3. The vector of claim 1, wherein the vector is retroviral and
wherein said coding sequence and said promoter are both positioned
between the 5' and 3' LTRs of the retroviral vector.
4. The vector of claim 3, wherein the retroviral vector is derived
from the avian leukosis virus (ALV), murine leukemia virus (MLV),
or lentivirus.
5. The vector of claim 1, further comprising a signal peptide
coding sequence which is operably linked to said coding sequence,
so that upon translation in a cell, the signal peptide will direct
secretion of the transgenic poultry derived interferon-.alpha. 2b
or erythropoietin expressed by the vector into the egg white of a
hard shell egg.
6. The vector of claim 1, further comprising a marker gene, wherein
said marker gene is operably linked to a promoter selected from the
group consisting of a cytomegalovirus (CMV) promoter, a MDOT
promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia
virus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter,
an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter,
an ovomucoid promoter, an ovomucin promoter, an ovotransferrin
promoter, and segments thereof.
7. A transgenic avian having a transgene in the genetic material of
its germ-line tissue, wherein the transgene comprises an exogenous
gene and a promoter in operational and positional relationship to
express transgenic poultry derived interferon-.alpha. 2b or
erythropoietin, wherein said exogenous gene is expressed in an
avian oviduct of said transgenic avian.
8. The transgenic avian of claim 7, wherein the promoter is
selected from the group consisting of a cytomegalovirus (CMV)
promoter, a MDOT promoter, a rous-sarcoma virus (RSV) promoter, a
murine leukemia virus (MLV) promoter, a mouse mammary tumor virus
(MMTV) promoter, an ovalbumin promoter, a lysozyme promoter, a
conalbumin promoter, an ovomucoid promoter, an ovomucin promoter,
an ovotransferrin promoter, and segments thereof.
9. The transgenic avian of claim 7, wherein the transgenic avian is
selected from the group consisting of chicken and turkey.
10. An egg of an avian species containing protein exogenous to the
avian species, wherein said protein is selected from the group
consisting of transgenic poultry derived interferon-.alpha. 2b and
erythropoietin.
11. A method for the stable introduction of an exogenous coding
sequence into the genome of an avian, comprising: introducing the
vector of claim 1 into avian embryonic blastodermal cells, wherein
the vector is randomly inserted into the avian genome.
12. A method for producing transgenic poultry derived
interferon-.alpha. 2b or erythropoietin protein in an avian
oviduct, comprising: providing a vector that comprises a coding
sequence and a promoter operably linked to said coding sequence,
wherein said promoter can effect expression of the coding sequence
in an avian oviduct; creating transgenic cells or tissue by
introducing said vector into avian embryonic blastodermal cells,
wherein the vector sequence is randomly inserted into the avian
genome; and deriving a mature transgenic avian from said transgenic
cells or tissue, wherein the transgenic avian expresses the
transgenic poultry derived interferon-.alpha. 2b or erythropoietin
protein in its oviduct and in its blood.
13. The method of claim 12, wherein said promoter is selected from
the group consisting of a cytomegalovirus (CMV) promoter, a MDOT
promoter, a rous-sarcoma virus (RSV) promoter, a murine leukemia
virus (MLV) promoter, a mouse mammary tumor virus (MMTV) promoter,
an ovalbumin promoter, a lysozyme promoter, a conalbumin promoter,
an ovomucoid promoter, an ovomucin promoter, an ovotransferrin
promoter, and segments thereof.
14. The method of claim 12, wherein the introduction of the vector
into the blastodermal cells is mediated by a retrovirus.
15. The method of claim 14, wherein the step of introducing said
vector to the embryonic blastodermal cells includes administering
helper cells to an embryonic blastoderm, wherein said helper cells
produce the retrovirus.
16. A method for producing an avian egg which contains transgenic
poultry derived interferon-.alpha. 2b or erythropoietin protein,
comprising: providing a vector that comprises a coding sequence and
a promoter operably linked to said coding sequence, wherein said
promoter can effect expression of the coding sequence in an avian
oviduct; creating transgenic cells or tissue by introducing said
vector into avian embryonic blastodermal cells, wherein the vector
sequence is randomly inserted into the avian genome; and deriving a
mature transgenic avian from said transgenic cells or tissue,
wherein the transgenic avian expresses the coding sequence in its
oviduct, and a resulting protein is secreted into the oviduct
lumen, so that the transgenic poultry derived interferon-.alpha. 2b
or erythropoietin protein is deposited into the egg white of a hard
shell egg.
17. An isolated polynucleotide comprising the optimized
polynucleotide sequence of human interferon-.alpha. 2b (SEQ ID NO:
1).
18. An isolated protein comprising the polypeptide sequence of
transgenic poultry derived interferon-.alpha. 2b, wherein said
protein is O-glycosylated at Thr-106 with Gal-NAcGal-, SA-
Gal-NAcGal-, 5Gal-Gal-NAcGal-, and 6
19. The isolated protein of claim 18, wherein Gal-NAcGal- is 20%,
SA-Gal-NAcGal- is 29%, 7is 9%, 8is 6%, Gal-Gal-NAcGal- is 7%, and
9
20. A pharmaceutical composition comprising the polypeptide
sequence of transgenic poultry derived interferon-.alpha. 2b,
wherein said protein is O-glycosylated at Thr-106 with Gal-NAcGal-,
SA-Gal-NAcGal-, 10Gal-Gal-NAcGal-, and 11
21. The isolated pharmaceutical composition of claim 20, wherein
Gal-NAcGal- is 20%, SA-Gal-NAcGal- is 29%, 12is 9%, 13is 6%,
Gal-Gal-NAcGal- is 7%, and 14
22. An isolated polynucleotide comprising the optimized
polynucleotide sequence of human erythropoietin (SEQ ID NO: 3).
23. An MDOT promoter, comprised of elements from the ovomucoid (MD)
and ovotransferrin (TO) promoters.
24. A vector comprising a first and second coding sequence and a
promoter in operational and positional relationship to said first
and second coding sequence to express said first and second coding
sequence in an avian oviduct; and an internal ribosome entry site
element positioned between the first and second coding sequence,
wherein said first coding sequence codes for protein X and said
second coding sequence codes for protein Y, and wherein said
protein X and protein Y are deposited into the egg white of a hard
shell egg.
25. The vector of claim 24, wherein protein X is a light chain (LC)
of a monoclonal antibody and protein Y is a heavy chain (HC) of a
monoclonal antibody.
26. The vector of claim 24, further comprising at least one
additional coding sequence and at least one additional internal
ribosome entry site element, wherein said internal ribosome entry
site element is further positioned between two coding sequences,
such that each coding sequence in the vector is separated from
another coding sequence by an internal ribosome entry site
element.
27. The vector of claim 24, wherein the protein encoded by the
second coding sequence is capable of providing post-translational
modification of the protein encoded by the first coding
sequence.
28. A method for producing an avian egg which contains a protein,
comprising: providing the vector of claim 24; creating transgenic
cells or tissue by introducing said vector into avian embryonic
blastodermal cells, wherein the vector sequence is randomly
inserted into the avian genome; and deriving a mature transgenic
avian from said transgenic cells or tissue, wherein the transgenic
avian expresses the coding sequences in its oviduct, and the
resulting protein is secreted into the oviduct lumen, so that the
protein is deposited into the egg white of a hard shell egg.
29. The method of claim 28, wherein the protein is a monoclonal
antibody.
30. A method for producing an avian egg which contains a protein,
comprising: providing the vector of claim 26; creating transgenic
cells or tissue by introducing said vector into avian embryonic
blastodermal cells, wherein the vector sequence is randomly
inserted into the avian genome; and deriving a mature transgenic
avian from said transgenic cells or tissue, wherein the transgenic
avian expresses the coding sequences in its oviduct, and the
resulting protein is secreted into the oviduct lumen, so that the
protein is deposited into the egg white of a hard shell egg.
31. The method of claim 30, wherein the protein is a monoclonal
antibody.
Description
[0001] This application is a continuation-in-part of copending U.S.
application Ser. No. 10/351,196, filed Jan. 24, 2003, which is a
continuation-in-part of copending U.S. application Ser. No.
09/173,864, filed Oct. 16, 1998, which claims the benefit of U.S.
Provisional Application Serial No. 60/062,172, filed Oct. 16, 1997,
all incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] a) Field of the Invention
[0003] The present invention relates to vectors and methods for the
introduction of exogenous genetic material into avian cells and the
expression of the exogenous genetic material in the cells. The
invention also relates to transgenic avian species, including
chicken and turkey, and to avian eggs which contain exogenous
protein.
[0004] b) Description of Related Art
[0005] Numerous natural and synthetic proteins are used in
diagnostic and therapeutic applications; many others are in
development or in clinical trials. Current methods of protein
production include isolation from natural sources and recombinant
production in bacterial and mammalian cells. Because of the
complexity and high cost of these methods of protein production,
however, efforts are underway to develop alternatives. For example,
methods for producing exogenous proteins in the milk of pigs,
sheep, goats, and cows have been reported. These approaches suffer
from several limitations, including long generation times between
founder and production transgenic herds, extensive husbandry and
veterinary costs, and variable levels of expression because of
position effects at the site of the transgene insertion in the
genome. Proteins are also being produced using milling and malting
processes from barley and rye. However, plant post-translational
modifications differ from vertebrate post-translational
modifications, which often has a critical effect on the function of
the exogenous proteins.
[0006] The Oviduct as a Bioreactor
[0007] Like tissue culture and mammary gland bioreactors, the avian
oviduct can also potentially serve as a bioreactor. Successful
methods of modifying avian genetic material such that high levels
of exogenous proteins are secreted in and packaged into eggs would
allow inexpensive production of large amounts of protein. Several
advantages of such an approach would be: a) short generation times
(24 weeks) and rapid establishment of transgenic flocks via
artificial insemination; b) readily scaled production by increasing
flock sizes to meet production needs; c) post-translational
modification of expressed proteins; 4) automated feeding and egg
collection; d) naturally sterile egg-whites; and e) reduced
processing costs due to the high concentration of protein in the
egg white.
[0008] The avian reproductive system, including that of the
chicken, is well described. The egg of the hen consists of several
layers which are secreted upon the yolk during its passage through
the oviduct. The production of an egg begins with formation of the
large yolk in the ovary of the hen. The unfertilized oocyte is then
positioned on top of the yolk sac. Upon ovulation or release of the
yolk from the ovary, the oocyte passes into the infundibulum of the
oviduct where it is fertilized if sperm are present. It then moves
into the magnum of the oviduct which is lined with tubular gland
cells. These cells secrete the egg-white proteins, including
ovalbumin, lysozyme, ovomucoid, conalbumin, and ovomucin, into the
lumen of the magnum where they are deposited onto the avian embryo
and yolk.
[0009] The ovalbumin gene encodes a 45 kD protein that is
specifically expressed in the tubular gland cells of the magnum of
the oviduct (Beato, Cell 56:335-344 (1989)). Ovalbumin is the most
abundant egg white protein, comprising over 50 percent of the total
protein produced by the tubular gland cells, or about 4 grams of
protein per large Grade A egg (Gilbert, "Egg albumen and its
formation" in Physiology and Biochemistry of the Domestic Fowl,
Bell and Freeman, eds., Academic Press, London, N.Y., pp.
1291-1329). The ovalbumin gene and over 20 kb of each flanking
region have been cloned and analyzed (Lai et al., Proc. Natl. Acad.
Sci. USA 75:2205-2209 (1978); Gannon et al., Nature 278:428-424
(1979); Roop et al., Cell 19:63-68 (1980); and Royal et al., Nature
279:125-132 (1975)).
[0010] Much attention has been paid to the regulation of the
ovalbumin gene. The gene responds to steroid hormones such as
estrogen, glucocorticoids, and progesterone, which induce the
accumulation of about 70,000 ovalbumin mRNA transcripts per tubular
gland cell in immature chicks and 100,000 ovalbumin mRNA
transcripts per tubular gland cell in the mature laying hen
(Palmiter, J. Biol. Chem. 248:8260-8270 (1973); Palmiter, Cell
4:189-197 (1975)). DNAse hypersensitivity analysis and
promoter-reporter gene assays in transfected tubular gland cells
defined a 7.4 kb region as containing sequences required for
ovalbumin gene expression. This 5' flanking region contains four
DNAse I-hypersensitive sites centered at -0.25, -0.8, -3.2, and
-6.0 kb from the transcription start site. These sites are called
HS-I, -II, -III, and -IV, respectively. These regions reflect
alterations in the chromatin structure and are specifically
correlated with ovalbumin gene expression in oviduct cells (Kaye et
al., EMBO 3:1137-1144 (1984)). Hypersensitivity of HS-II and -III
are estrogen-induced, supporting a role for these regions in
hormone-induction of ovalbumin gene expression.
[0011] HS-I and HS-II are both required for steroid induction of
ovalbumin gene transcription, and a 1.4 kb portion of the 5' region
that includes these elements is sufficient to drive
steroid-dependent ovalbumin expression in explanted tubular gland
cells (Sanders and McKnight, Biochemistry 27: 6550-6557 (1988)).
HS-I is termed the negative-response element ("NRE") because it
contains several negative regulatory elements which repress
ovalbumin expression in the absence of hormones (Haekers et al.,
Mol. Endo. 9:1113-1126 (1995)). Protein factors bind these
elements, including some factors only found in oviduct nuclei
suggesting a role in tissue-specific expression. HS-II is termed
the steroid-dependent response element ("SDRE") because it is
required to promote steroid induction of transcription. It binds a
protein or protein complex known as Chirp-I. Chirp-I is induced by
estrogen and turns over rapidly in the presence of cyclohexamide
(Dean et al., Mol. Cell. Biol. 16:2015-2024 (1996)). Experiments
using an explanted tubular gland cell culture system defined an
additional set of factors that bind SDRE in a steroid-dependent
manner, including a NF.kappa.B-like factor (Nordstrom et al., J.
Biol. Chem. 268:13193-13202 (1993); Schweers and Sanders, J. Biol.
Chem. 266: 10490-10497 (1991)).
[0012] Less is known about the function of HS-III and -IV. HS-III
contains a functional estrogen response element, and confers
estrogen inducibility to either the ovalbumin proximal promoter or
a heterologous promoter when co-transfected into HeLa cells with an
estrogen receptor cDNA. These data imply that HS-III may play a
functional role in the overall regulation of the ovalbumin gene.
Little is known about the function of HS-IV, except that it does
not contain a functional estrogen-response element (Kato et al.,
Cell 68: 731-742 (1992)).
[0013] There has been much interest in modifying eukaryotic genomes
by introducing foreign genetic material and/or by disrupting
specific genes. Certain eukaryotic cells may prove to be superior
hosts for the production of exogenous eukaryotic proteins. The
introduction of genes encoding certain proteins also allows for the
creation of new phenotypes which could have increased economic
value. In addition, some genetically-caused disease states may be
cured by the introduction of a foreign gene that allows the
genetically defective cells to express the protein that it can
otherwise not produce. Finally, modification of animal genomes by
insertion or removal of genetic material permits basic studies of
gene function, and ultimately may permit the introduction of genes
that could be used to cure disease states, or result in improved
animal phenotypes.
[0014] Transgenic Animals
[0015] Transgenesis has been accomplished in mammals by several
different methods. First, in mammals including the mouse, pig,
goat, sheep and cow, a transgene is microinjected into the
pronucleus of a fertilized egg, which is then placed in the uterus
of a foster mother where it gives rise to a founder animal carrying
the transgene in its germline. The transgene is engineered to carry
a promoter with specific regulatory sequences directing the
expression of the foreign protein to a particular cell type. Since
the transgene inserts randomly into the genome, position effects at
the site of the transgene's insertion into the genome may variably
cause decreased levels of transgene expression. This approach also
requires characterization of the promoter such that sequences
necessary to direct expression of the transgene in the desired cell
type are defined and included in the transgene vector (Hogan et al.
Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory, NY
(1988)).
[0016] A second method for effecting animal transgenesis is
targeted gene disruption, in which a targeting vector bearing
sequences of the target gene flanking a selectable marker gene is
introduced into embryonic stem ("ES") cells. Via homologous
recombination, the targeting vector replaces the target gene
sequences at the chromosomal locus or inserts into interior
sequences preventing expression of the target gene product. Clones
of ES cells bearing the appropriately disrupted gene are selected
and then injected into early stage blastocysts generating chimeric
founder animals, some of which bear the transgene in the germ line.
In the case where the transgene deletes the target locus, it
replaces the target locus with foreign DNA borne in the transgene
vector, which consists of DNA encoding a selectable marker useful
for detecting transfected ES cells in culture and may additionally
contain DNA sequences encoding a foreign protein which is then
inserted in place of the deleted gene such that the target gene
promoter drives expression of the foreign gene (U.S. Pat. Nos.
5,464,764 and 5,487,992 (M. P. Capecchi and K. R. Thomas)). This
approach suffers from the limitation that ES cells are unavailable
in many mammals, including goats, cows, sheep and pigs.
Furthermore, this method is not useful when the deleted gene is
required for survival or proper development of the organism or cell
type.
[0017] Recent developments in avian transgenesis have allowed the
modification of avian genomes. Germ-line transgenic chickens may be
produced by injecting replication-defective retrovirus into the
subgerminal cavity of chick blastoderms in freshly laid eggs (U.S.
Pat. No. 5,162,215; Bosselman et al., Science 243:533-534 (1989);
Thoraval et al., Transgenic Research 4:369-36 (1995)). The
retroviral nucleic acid carrying a foreign gene randomly inserts
into a chromosome of the embryonic cells, generating transgenic
animals, some of which bear the transgene in their germ line. Use
of insulator elements inserted at the 5' or 3' region of the fused
gene construct to overcome position effects at the site of
insertion has been described (Chim et al., Cell 74:504-514
(1993)).
[0018] In another approach, a transgene has been microinjected into
the germinal disc of a fertilized egg to produce a stable
transgenic founder avian that passes the gene to the F1 generation
(Love et al. Bio/Technology 12:60-63 (1994)). However, this method
has several disadvantages. Hens must be sacrificed in order to
collect the fertilized egg, the fraction of transgenic founders is
low, and injected eggs require labor intensive in vitro culture in
surrogate shells.
[0019] In another approach, blastodermal cells containing
presumptive primordial germ cells ("PGCs") are excised from donor
eggs, transfected with a transgene and introduced into the
subgerminal cavity of recipient embryos. The transfected donor
cells are incorporated into the recipient embryos generating
transgenic embryos, some of which are expected to bear the
transgene in the germ line. The transgene inserts in random
chromosomal sites by nonhomologous recombination. However, no
transgenic founder avians have yet been generated by this
method.
[0020] Lui, Poult. Sci. 68:999-1010 (1995), used a targeting vector
containing flanking DNA sequences of the vitellogenin gene to
delete part of the resident gene in chicken blastodermal cells in
culture. However, it has not been demonstrated that these cells can
contribute to the germ line and thus produce a transgenic embryo.
In addition, this method is not useful when the deleted gene is
required for survival or proper development of the organism or cell
type.
[0021] Thus, it can be seen that there is a need for a method of
introducing foreign DNA, operably linked to a suitable promoter,
into the avian genome such that efficient expression of an
exogenous gene can be achieved. Furthermore, there exists a need to
create germ-line modified transgenic avians which express exogenous
genes in their oviducts and secrete the expressed exogenous
proteins into their eggs.
[0022] Interferon
[0023] When interferon was discovered in 1957, it was hailed as a
significant antiviral agent. In the late 1970s, interferon became
associated with recombinant gene technology. Today, interferon is a
symbol of the complexity of the biological processes of cancer and
the value of endurance and persistence in tackling this
complexity.
[0024] The abnormal genes that cause cancer comprise at least three
types: Firstly, there are the oncogenes, which, when altered,
encourage the abnormal growth and division that characterize
cancer. Secondly, there are the tumor suppressor genes, which, when
altered, fail to control this abnormal growth and division.
Thirdly, there are the DNA repair genes, which, when altered, fail
to repair mutations that can lead to cancer. Researchers speculate
that there are about 30 to 40 tumor suppressor genes in the body,
each of which produces a protein. These proteins may be controlled
by "master" tumor suppressor proteins such as Rb (for
retinoblastoma, with which it was first associated) and p53
(associated with many different tumors). Evidence from the
laboratory suggests that returning just one of these tumor
suppressor genes to its normal function can appreciably reduce the
aggressiveness of the malignancy.
[0025] Scientists became intrigued by interferon when it was
discovered that interferon can inhibit cell growth. Further,
interferon was found to have certain positive effects on the immune
system. It is now considered analogous to a tumor suppressor
protein: it inhibits the growth of cells, particularly malignant
cells; it blocks the effects of many oncogenes and growth factors;
and unlike other biological agents, it inhibits cell motility which
is critical to the process of metastasis.
[0026] Intercellular communication is dependent on the proper
functioning of all the structural components of the tissue through
which the messages are conveyed: the matrix, the cell membrane, the
cytoskeleton, and the cell itself. In cancer, the communication
network between cells is disrupted. If the cytoskeleton is
disrupted, the messages don't get through to the nucleus and the
nucleus begins to function abnormally. Since the nucleus is the
site where the oncogenes or tumor suppressor genes get switched on
or off, this abnormal functioning can lead to malignancy. When this
happens, the cells start growing irregularly and do not
differentiate. They may also start to move and disrupt other cells.
It is believed that interferon, probably in concert with other
extracellular and cellular substances, restores the balance and
homeostasis, making sure the messages get through properly.
Interferon stops growth, stops motility, and enhances the ability
of the cell, through adhesion molecules, to respond to its
environment. It also corrects defects and injuries in the
cytoskeleton. Interferon has been found to block angiogenesis, the
initial step in the formation of new blood vessels that is
essential to the growth of malignancies. Moreover, it blocks
fibrosis, a response to injury that stimulates many different kinds
of cells and promotes cell growth (Kathryn L. Hale, Oncolog,
Interferon: The Evolution of a Biological Therapy, Taking a New
Look at Cytokine Biology).
[0027] Interferon is produced by animal cells when they are invaded
by viruses and is released into the bloodstream or intercellular
fluid to induce healthy cells to manufacture an enzyme that
counters the infection. For many years the supply of human
interferon for research was limited by costly extraction
techniques. In 1980, however, the protein became available in
greater quantities through genetic engineering (i.e., recombinant
forms of the protein). Scientists also determined that the body
makes three distinct types of interferon, referred to as
.alpha.-(alpha), .beta.-(beta), and .gamma.-(gamma) interferon.
Interferons were first thought to be highly species-specific, but
it is now known that individual interferons may have different
ranges of activity in other species. Alpha interferon (.alpha.-IFN)
has been approved for therapeutic use against hairy-cell leukemia
and hepatitis C. .alpha.-IFN has also been found effective against
chronic hepatitis B, a major cause of liver cancer and cirrhosis,
as well as for treatment of genital warts and some rarer cancers of
blood and bone marrow. Nasal sprays containing .alpha.-IFN provide
some protection against colds caused by rhinoviruses. Human
.alpha.-IFN belongs to a family of extra-cellular signaling
proteins with antiviral, antiproliferating and immunomodulatory
activities. IFN-.alpha. proteins are encoded by a multigene family
which includes 13 genes clustered on the human chromosome 9. Most
of the IFN-.alpha. genes are expressed at the mRNA level in
leukocytes induced by Sendai virus. Further, it has been shown that
at least nine different sub-types are also produced at the protein
level. The biological significance of the expression of several
similar IFN-.alpha. proteins is not known, however, it is believed
that they have quantitatively distinct patterns of antiviral,
growth inhibitory and killer-cell-stimulatory activities.
Currently, two IFN-.alpha. variants, IFN-.alpha. 2a and IFN-.alpha.
2b, are mass produced in Escherichia coli by recombinant technology
and marketed as drugs. Unlike natural IFN-.alpha., these
recombinant IFN-.alpha. products have been shown to be immunogenic
in some patients, which could be due to unnatural forms of
IFN-.alpha. proteins. Thus, for the development of IFN-.alpha.
drugs it is necessary to not only identify the IFN-.alpha. subtypes
and variants expressed in normal human leukocytes, but also to
characterize their possible post-translational modifications (Nyman
et al. (1998) Eur. J. Biochem. 253:485-493).
[0028] Nyman et al. (supra) studied the glycosylation of natural
human IFN-.alpha.. They found that two out of nine of the subtypes
produced by leukocytes after a Sendai-virus induction were found to
be glycosylated, namely IFN-.alpha. 14c and IFN-.alpha. 2b, which
is consistent with earlier studies. IFN-.alpha. 14 is the only
IFN-.alpha. subtype with potential N-glycosylation sites, Asn2 and
Asn72, but only Asn72 is actually glycosylated. IFN-.alpha. 2 is
O-glycosylated at Threonine 106 (Thr106). Interestingly, no other
IFN-.alpha. subtype contains Thr at this position. In this study,
Nyman et al. liberated and isolated the oligosaccharide chains and
analyzed their structures by mass spectrometry and specific
glycosidase digestions. Both IFN-.alpha. 2b and IFN-.alpha. 14c
resolved into three peaks in reversed-phase high performance liquid
chromatography (RP-HPLC). Electrospray ionization mass spectrometry
(ESI-MS) analysis of IFN-.alpha. 2b fractions from RP-HPLC revealed
differences in their molecular masses, suggesting that these
represent different glycoforms. This was confirmed by
masspectrometric analysis of the liberated O-glycans of each
fraction. IFN-.alpha. 2b was estimated to contain about 20% of the
core type-2 pentasaccharide, and about 50% of disialylated and 30%
of monosialylated core type-1 glycans. Nyman et al.'s data agrees
with previous partial characterization of IFN-.alpha. 2b
glycosylation (Adolf et al. (1991) Biochem. J. 276:511-518). The
role of glycosylation in IFN-.alpha. 14c and IFN-.alpha. 2b is not
clearly established. According to Nyman et al. (supra), the
carbohydrate chains are not essential for the biological activity,
but glycosylation may have an effect on the pharmacokinetics and
stability of the proteins.
[0029] There are at least 15 functional genes in the human genome
that code for proteins of the IFN-.alpha. family. The amino acid
sequence similarities are generally in the region of about 90%,
thus, these molecules are closely related in structure. IFN-.alpha.
proteins contain 166 amino acids (with the exception of IFN-.alpha.
2, which has 165 amino acids) and characteristically contain four
conserved cysteine residues which form two disulfide bridges.
IFN-.alpha. species are slightly acidic in character and lack a
recognition site for asparagine-linked glycosylation (with the
exception of IFN-.alpha. 14 which does contain a recognition site
for asparagine-linked glycosylation). Three variants of IFN-.alpha.
2, differing in their amino acids at positions 23 and 34, are
known: IFN-.alpha. 2a (Lys-23, His-34); IFN-.alpha. 2b (Arg-23,
His-34); and IFN-.alpha. 2c (Arg-23, Arg-34). Two other human IFN
species, namely IFN-.omega.1 and IFN-.beta. are N-glycosylated and
are more distantly related to IFN-.alpha.. IFN-.alpha., -.beta. and
-.omega., collectively referred to as class I IFNs, bind to the
same high affinity cell membrane receptor (Adolf et al. (1991)
Biochem. J. 276:511-518).
[0030] Adolf et al. (supra) used the specificity of a monoclonal
antibody for the isolation of natural IFN-.alpha. 2 from human
leukocyte IFN. They obtained a 95% pure protein through
immunoaffinity chromatography which confirmed the expected
antiviral activity of IFN-.alpha. 2. Analysis of natural
IFN-.alpha. 2 by reverse-phase HPLC, showed that the natural
protein can be resolved into two components, both more hydrophilic
than E. coli-derived IFN-.alpha. 2. SDS/PAGE revealed that the
protein is also heterogeneous in molecular mass, resulting in three
bands, all of them with lower electrophoretic mobility than the
equivalent E. coli-derived protein.
[0031] Adolf et al. (supra) also speculated that natural
IFN-.alpha. 2 carries O-linked carbohydrate residues. Their
hypothesis was confirmed by cleavage of the putative
peptide-carbohydrate bond with alkali; the resulting protein was
homogeneous and showed the same molecular mass as the recombinant
protein. Further comparison of natural and recombinant proteins
after proteolytic cleavage, followed by separation and analysis of
the resulting fragments, allowed them to define a candidate
glycopeptide. Sequence analysis of this peptide identified Thr-106
as the O-glycosylation site. A comparison of the amino acid
sequences of all published IFN-.alpha. 2 species revealed that this
threonine residue is unique to IFN-.alpha. 2. Glycine, isoleucine
or glutamic acid are present at the corresponding position (107) in
all other proteins.
[0032] Preparations of IFN-.alpha. 2 produced in E. coli are devoid
of O-glycosylation and have been registered as drugs in many
countries. However, the immunogenicity of therapeutically applied
E. coli-derived IFN-.alpha. 2 might be affected by the lack of
glycosylation. Studies have shown that four out of sixteen patients
receiving recombinant human granulocyte-macrophage
colony-stimulating factor produced in yeast developed antibodies to
this protein. Interestingly, these antibodies were found to react
with epitopes that in the endogenous granulocyte-macrophage
colony-stimulating factor are protected by O-linked glycosylation,
but which are exposed in the recombinant factor (Adolf et al.,
supra).
[0033] Similarly, induction of antibodies to recombinant E.
coli-derived IFN-.alpha. 2 after prolonged treatment of patients
has been described and it has been speculated that natural
IFN-.alpha. 2 may be less immunogenic than the recombinant
IFN-.alpha. 2 proteins (Galton et al. (1989) Lancet 2:572-573).
SUMMARY OF THE INVENTION
[0034] This invention provides vectors and methods for the stable
introduction of exogenous nucleic acid sequences into the genome of
avians in order to express the exogenous sequences to alter the
phenotype of the avians or to produce desired proteins. In
particular, transgenic avians are produced which express exogenous
sequences in their oviducts and which deposit exogenous proteins
into their eggs. Avian eggs that contain exogenous proteins are
encompassed by this invention. The instant invention further
provides novel forms of interferon and erythropoietin which are
efficiently expressed in the oviduct of transgenic avians and
deposited into avian eggs.
[0035] One aspect of the present invention provides methods for
producing exogenous proteins in specific tissues of avians.
Exogenous proteins may be expressed in the oviduct, blood and/or
other cells and tissues of the avian. Transgenes are introduced
into embryonic blastodermal cells, preferably near stage X, to
produce a transgenic avian, such that the protein of interest is
expressed in the tubular gland cells of the magnum of the oviduct,
secreted into the lumen, and deposited into the egg white of a hard
shell egg. A transgenic avian so produced carries the transgene in
its germ line. The exogenous genes can therefore be transmitted to
avians by both artificial introduction of the exogenous gene into
avian embryonic cells, and by the transmission of the exogenous
gene to the avian's offspring stably in a Mendelian fashion.
[0036] The present invention encompasses a method of producing an
exogenous protein in an avian oviduct. The method comprises as a
first step providing a vector that contains a coding sequence and a
promoter operably linked to the coding sequence, so that the
promoter can effect expression of the nucleic acid in the avian
oviduct. Next, transgenic cells and/or tissues are created, wherein
the vector is introduced into avian embryonic blastodermal cells,
either freshly isolated, in culture, or in an embryo, so that the
vector sequence is randomly inserted into the avian genome.
Finally, a mature transgenic avian which expresses the exogenous
protein in its oviduct is derived from the transgenic cells and/or
tissue. This method can also be used to produce an avian egg which
contains exogenous protein when the exogenous protein that is
expressed in the oviduct is also secreted into the oviduct lumen
and deposited into the egg white of a hard shell egg.
[0037] In one aspect, the production of a transgenic bird by random
chromosomal insertion of a vector into its avian genome may
optionally involve DNA transfection of embryonic blastodermal cells
which are then injected into the subgerminal cavity beneath a
recipient blastoderm. The vector used in such a method has a
promoter which is fused to an exogenous coding sequence and directs
expression of the coding sequence in the tubular gland cells of the
oviduct.
[0038] In another aspect of the invention, a random chromosomal
insertion and the production of a transgenic avian is accomplished
by transduction of embryonic blastodermal cells with
replication-defective or replication-competent retroviral particles
carrying the transgene genetic code between the 5' and 3' LTRs of
the retroviral rector. For instance, an avian leukosis virus (ALV)
retroviral vector or a murine leukemia virus (MLV) retroviral
vector may be used which comprises a modified pNLB plasmid
containing an exogenous gene that is inserted downstream of a
segment of a promoter region. An RNA copy of the modified
retroviral vector, packaged into viral particles, is used to infect
embryonic blastoderms which develop into transgenic avians.
Alternatively, helper cells which produce the retroviral
transducing particles are delivered to the embryonic
blastoderm.
[0039] Another aspect of the invention provides a vector which
includes a coding sequence and a promoter in operational and
positional relationship such that the coding sequence is expressed
in an avian oviduct. The vector includes, but is not limited to, an
avian leukosis virus (ALV) retroviral vector, a murine leukemia
virus (MLV) retroviral vector, and a lentivirus vector. The
promoter is sufficient for effecting expression of the coding
sequence in the avian oviduct. The coding sequence codes for an
exogenous protein which is deposited into the egg white of a hard
shell egg. As such, the coding sequence codes for exogenous
proteins such as transgenic poultry derived interferon-.alpha. 2b
(TPD IFN-.alpha. 2b) or transgenic poultry derived erythropoietin
(TPD EPO). The vector used in the methods of the invention contains
a promoter which is particularly suited for expression of exogenous
proteins in avians and their eggs. As such, expression of the
exogenous coding sequence occurs in the oviduct and blood of the
transgenic avian and in the egg white of its avian egg. The
promoter includes, but is not limited to, a cytomegalovirus (CMV)
promoter, a MDOT promoter, a rous-sarcoma virus (RSV) promoter, a
murine leukemia virus (MLV) promoter, a mouse mammary tumor virus
(MMTV) promoter, an ovalbumin promoter, a lysozyme promoter, a
conalbumin promoter, an ovomucoid promoter, an ovomucin promoter,
and an ovotransferrin promoter. Optionally, the promoter may be a
segment of at least one promoter region, such as a segment of the
ovalbumin-, lysozyme-, conalbumin-, ovomucoid-, ovomucin-, and
ovotransferrin promoter region.
[0040] One aspect of the invention involves truncating the
ovalbumin promoter and/or condensing the critical regulatory
elements of the ovalbumin promoter so that it retains sequences
required for expression in the tubular gland cells of the magnum of
the oviduct, while being small enough that it can be readily
incorporated into vectors. For instance, a segment of the ovalbumin
promoter region may be used. This segment comprises the 5'-flanking
region of the ovalbumin gene. The total length of the ovalbumin
promoter segment may be from about 0.88 kb to about 7.4 kb in
length, and is preferably from about 0.88 kb to about 1.4 kb in
length. The segment preferably includes both the steroid-dependent
regulatory element and the negative regulatory element of the
ovalbumin gene. The segment optionally also includes residues from
the 5'untranslated region (5'UTR) of the ovalbumin gene.
Alternatively, the promoter may be a segment of the promoter region
of the lysozyme-, conalbumin-, ovomucin-, ovomucoid- and
ovotransferrin genes. An example of such a promoter is the
synthetic MDOT promoter which is comprised of elements from the
ovomucoid (MD) and ovotransferrin (TO) promoter.
[0041] In another aspect of the invention, the vectors integrated
into the avian genome contain constitutive promoters which are
operably linked to the exogenous coding sequence (e.g.,
cytomegalovirus (CMV) promoter, rous-sarcoma virus (RSV) promoter,
and a murine leukemia virus (MLV) promoter. Alternatively, a
non-constitutive promoter such as a mouse mammary tumor virus
(MMTV) promoter may be used.
[0042] Other aspects of the invention provide for transgenic avians
which carry a transgene in the genetic material of their germ-line
tissue. More specifically, the transgene includes an exogenous gene
and a promoter in operational and positional relationship to
express the exogenous gene. The exogenous gene is expressed in the
avian oviduct and in the blood of the transgenic avian. The
exogenous gene codes for exogenous proteins such as TPD IFN-.alpha.
2b or TPD EPO. The exogenous protein is deposited into the egg
white of a hard shell egg.
[0043] Another aspect of the invention provides for an avian egg
which contains protein exogenous to the avian species. Use of the
invention allows for expression of exogenous proteins in oviduct
cells with secretion of the proteins into the lumen of the oviduct
magnum and deposition into the egg white of the avian egg. Proteins
packaged into eggs may be present in quantities of up to one gram
or more per egg. The exogenous protein includes, but is not limited
to, TPD IFN-.alpha. 2b and TPD EPO.
[0044] Still another aspect of the invention provides an isolated
polynucleotide sequence comprising the optimized coding sequence of
human interferon-.alpha. 2b (IFN-.alpha. 2b), i.e., recombinant
transgenic poultry derived interferon-.alpha. 2b coding sequence
which codes for transgenic poultry derived interferon-.alpha. 2b
(TPD IFN-.alpha. 2b). The invention also encompasses an isolated
protein comprising the polypeptide sequence of TPD IFN-.alpha. 2b,
wherein the protein is O-glycosylated at Thr-106 with
N-Acetyl-Galactosamine, Galactose, N-Acetyl-Glucosamine, Sialic
acid, and combinations thereof.
[0045] The invention further contemplates a pharmaceutical
composition comprising the polypeptide sequence of TPD IFN-.alpha.
2b, wherein the protein is O-glycosylated at Thr-106 with
N-Acetyl-Galactosamine, Galactose, N-Acetyl-Glucosamine, Sialic
acid, and combinations thereof.
[0046] Another aspect of the invention provides an isolated
polynucleotide sequence comprising the optimized coding sequence of
human erythropoietin (EPO), i.e., recombinant transgenic poultry
derived erythropoietin coding sequence which codes for transgenic
poultry derived erythropoietin (TPD EPO).
[0047] Yet another aspect of the invention provides for a vector
comprising a first and second coding sequence and a promoter in
operational and positional relationship to the first and second
coding sequence to express the first and second coding sequence in
an avian oviduct. The vector further includes an internal ribosome
entry site (IRES) element positioned between the first and second
coding sequence, wherein the first coding sequence codes for
protein X and the second coding sequence codes for protein Y, and
wherein protein X and protein Y are deposited into the egg white of
a hard shell egg. For example, protein X may be a light chain (LC)
of a monoclonal antibody and protein Y may be a heavy chain (HC) of
a monoclonal antibody. Alternatively, the protein encoded by the
second coding sequence (e.g., enzyme) may be capable of providing
post-translational modification of the protein encoded by the first
coding sequence. The vector optionally includes additional coding
sequences and additional IRES elements, such that each coding
sequence in the vector is separated from another coding sequence by
an IRES element.
[0048] The invention also contemplates methods of producing an
avian egg which contains proteins such as monoclonal antibodies,
enzymes, or other proteins. Such a method includes providing a
vector with a promoter, coding sequences, and at least one IRES
element; creating transgenic cells or tissue by introducing the
vector into avian embryonic blastodermal cells, wherein the vector
sequence is randomly inserted into the avian genome; and deriving a
mature transgenic avian from the transgenic cells or tissue. The
transgenic avian so derived expresses the coding sequences in its
oviduct, and the resulting protein is secreted into the oviduct
lumen, so that the protein is deposited into the egg white of a
hard shell egg.
BRIEF DESCRIPTION OF THE DRAWINGS
[0049] FIGS. 1A and 1B illustrate ovalbumin promoter expression
vectors comprising ovalbumin promoter segments and a coding
sequence, gene X, which encodes an exogenous protein X. X
represents any exogenous gene or exogenous protein of interest.
[0050] FIGS. 2A, 2B, 2C and 2D illustrate retroviral vectors of the
invention comprising an ovalbumin promoter and a coding sequence,
gene X, encoding an exogenous protein X. X represents any exogenous
gene or exogenous protein of interest.
[0051] FIG. 2E illustrates a method of amplifying an exogenous gene
for insertion into the vectors of 2A and 2B.
[0052] FIG. 2F illustrates a retroviral vector comprising an
ovalbumin promoter controlling expression of a coding sequence,
gene X, and an internal ribosome entry site (IRES) element enabling
expression of a second coding sequence, gene Y. X and Y represent
any gene of interest.
[0053] FIGS. 3A and 3B show schematic representations of the
ALV-derived vectors pNLB and pNLB-CMV-BL, respectively. Because NLB
has not been sequenced in its entirety, measurements in bp (base
pair) are estimated from published data (Cosset et al., 1991;
Thoraval et al., 1995) and data discussed herein. The vectors are
both shown as they would appear while integrated into the chicken
genome.
[0054] FIGS. 4A and 4B show the amount of .beta.-lactamase
(lactamase) in the blood serum of chimeric and transgenic chickens.
In FIG. 4A the concentration of bioactive lactamase in the serum of
G0 chickens transduced with the NLB-CMV-BL transgene was measured
at 8 month post-hatch. The generation, sex and wing band numbers
are indicated. Lactamase serum concentrations were measured for G1
transgenic chickens at 6 to 7 months post-hatch. Arrows indicate G1
chickens bred from rooster 2395. In FIG. 4B the lactamase serum
concentration was measured for G1 and G2 transgenic chickens.
Arrows indicate G2s bred from hen 5657 or rooster 4133. Samples
from chickens 4133, 5308, and 5657 are the same as those in FIG.
4A. Samples from G2 birds bred from 5657 were collected at 3 to 60
days post-hatch. Samples from G2 birds bred from 4133 were
collected at 3 month post-hatch.
[0055] FIG. 5 shows the pedigree of chickens bearing the transgenic
loci harbored by hen 5657 (FIG. 5A) or rooster 4133 (FIG. 5B). 2395
was a rooster that carried multiple transgenic loci. 2395 was bred
to a non-transgenic hen, yielding 3 offspring each carrying the
transgene in a unique position of the chicken genome. For
simplicity, transgenic progeny for which expression data were not
shown as well as non-transgenic progeny were omitted from the
pedigree. Band numbers are indicated by the following symbols:
.smallcircle. hen; .quadrature. rooster; .circle-solid. hen
carrying the NLB-CMV-BL transgene; .box-solid. rooster carrying the
NLB-CMV-BL transgene.
[0056] FIG. 6 shows .beta.-lactamase (lactamase) in the egg white
of hen 5657 and her offspring. In FIG. 6A egg white from hen 5657
and her transgenic offspring were assayed for active lactamase. The
control is from untreated hens and clutchmate is a non-transgenic
G2 bred from hen 5657. Eggs were collected in March 2000. Arrows
indicate G2s bred from hen 5657. In FIG. 6B egg white samples from
G2 transgenic hens carrying one copy of the transgene (hemizygous)
were compared with that of G3 hen 6978 which harbored two copies
(homozygous). Eggs were collected in February 2001. The generation
and wing band numbers are indicated to the left.
[0057] FIG. 7 shows .beta.-lactamase (lactamase) in the eggs of G2
and G3 hens bred from rooster 4133. In FIG. 7A egg whites from four
representative hemizygous transgenic hens bred from rooster 4133
were assayed for active lactamase. Eggs were collected in October
1999, March, 2000 and February 2001 and a minimum of 4 eggs per hen
were assayed one month after each set was collected. The control
represents egg white from untreated hens. Band numbers are
indicated to the left. The average of the 4 hens for each period is
calculated. In FIG. 7B egg white from hemizygous G2 transgenic hens
were compared with that of hemizygous and homozygous transgenic G3
hens. The eggs were collected in February 2001. The generation and
transgene copy number are displayed in the data bar for each hen.
The average concentration for hens carrying one or two copies is at
the bottom of the chart.
[0058] FIGS. 8A and 8B show the pNLB-CMV-IFN vector for expressing
IFN-.alpha. 2b in chickens; and the pNLB-MDOT-EPO vector used for
expressing erythropoietin (EPO) in chickens, respectively.
[0059] FIG. 9 depicts the novel glycosylation pattern of transgenic
poultry derived interferon-.alpha. 2b (TPD IFN-.alpha. 2b),
including all 6 bands.
[0060] FIG. 10 shows the comparison of human peripheral blood
leukocyte derived interferon-.alpha. 2b (PBL IFN-.alpha. 2b or
natural hIFN) and transgenic poultry derived interferon-.alpha. 2b
(TPD IFN-.alpha. 2b or egg white hIFN).
[0061] FIG. 11A depicts the synthetic nucleic acid sequence (cDNA,
residues 1-498) of optimized human interferon-.alpha. 2b
(IFN-.alpha. 2b), i.e., recombinant TPD IFN-.alpha. 2b (SEQ ID NO:
1). FIG. 11B depicts the synthetic amino acid sequence (residues
1-165) of transgenic poultry derived interferon-.alpha. 2b (TPD
IFN-.alpha. 2b) (SEQ ID NO: 2).
[0062] FIG. 12A depicts the synthetic nucleic acid sequence (cDNA,
residues 1-579) of optimized human erythropoietin (EPO) i.e.,
recombinant TPD EPO (SEQ ID NO: 3). FIG. 12B depicts the synthetic
amino acid sequence (residues 1-193) of transgenic poultry derived
erythropoietin (TPD EPO) (SEQ ID NO: 4). (For natural human EPO see
also NCBI Accession Number NP.sub.--000790).
[0063] FIG. 13 shows the synthetic MDOT promoter linked to the
IFN-MM CDS. The MDOT promoter contains elements from the chicken
ovomucoid gene (ovomucoid promoter) ranging from -435 to -166 bp
(see NCBI Accession Number J00894) and the chicken conalbumin gene
(ovotransferrin promoter) ranging from -251 to +29 bp (see NCBI
Accession Numbers Y00497, M11862 and X01205).
[0064] FIG. 14 provides a summary of the major egg white
proteins.
[0065] FIGS. 15A and 15D show the pCMV-LC-emcvIRES-HC vector,
wherein the light chain (LC) and heavy chain (HC) of a human
monoclonal antibody were expressed from this single vector by
placement of an IRES from the encephalomyocarditis virus (EMCV) in
order to test for expression of monoclonal antibodies. In
comparison, FIGS. 15B and 15C show the separate vectors pCMV-HC and
pCMV-LC, respectively, wherein these vectors were also used to test
for expression of monoclonal antibodies.
DETAILED DESCRIPTION OF THE INVENTION
[0066] a) Definitions and General Parameters
[0067] The following definitions are set forth to illustrate and
define the meaning and scope of the various terms used to describe
the invention herein.
[0068] A "nucleic acid or polynucleotide sequence" includes, but is
not limited to, eukaryotic mRNA, cDNA, genomic DNA, and synthetic
DNA and RNA sequences, comprising the natural nucleoside bases
adenine, guanine, cytosine, thymidine, and uracil. The term also
encompasses sequences having one or more modified bases.
[0069] A "coding sequence" or "open reading frame" refers to a
polynucleotide or nucleic acid sequence which can be transcribed
and translated (in the case of DNA) or translated (in the case of
mRNA) into a polypeptide in vitro or in vivo when placed under the
control of appropriate regulatory sequences. The boundaries of the
coding sequence are determined by a translation start codon at the
5' (amino) terminus and a translation stop codon at the 3'
(carboxy) terminus. A transcription termination sequence will
usually be located 3' to the coding sequence. A coding sequence may
be flanked on the 5' and/or 3' ends by untranslated regions.
[0070] "Exon" refers to that part of a gene which, when transcribed
into a nuclear transcript, is "expressed" in the cytoplasmic mRNA
after removal of the introns or intervening sequences by nuclear
splicing.
[0071] Nucleic acid "control sequences" or "regulatory sequences"
refer to promoter sequences, translational start and stop codons,
ribosome binding sites, polyadenylation signals, transcription
termination sequences, upstream regulatory domains, enhancers, and
the like, as necessary and sufficient for the transcription and
translation of a given coding sequence in a defined host cell.
Examples of control sequences suitable for eukaryotic cells are
promoters, polyadenylation signals, and enhancers. All of these
control sequences need not be present in a recombinant vector so
long as those necessary and sufficient for the transcription and
translation of the desired gene are present.
[0072] "Operably or operatively linked" refers to the configuration
of the coding and control sequences so as to perform the desired
function. Thus, control sequences operably linked to a coding
sequence are capable of effecting the expression of the coding
sequence. A coding sequence is operably linked to or under the
control of transcriptional regulatory regions in a cell when DNA
polymerase will bind the promoter sequence and transcribe the
coding sequence into mRNA that can be translated into the encoded
protein. The control sequences need not be contiguous with the
coding sequence, so long as they function to direct the expression
thereof. Thus, for example, intervening untranslated yet
transcribed sequences can be present between a promoter sequence
and the coding sequence and the promoter sequence can still be
considered "operably linked" to the coding sequence.
[0073] The terms "heterologous" and "exogenous" as they relate to
nucleic acid sequences such as coding sequences and control
sequences, denote sequences that are not normally associated with a
region of a recombinant construct or with a particular chromosomal
locus, and/or are not normally associated with a particular cell.
Thus, an "exogenous" region of a nucleic acid construct is an
identifiable segment of nucleic acid within or attached to another
nucleic acid molecule that is not found in association with the
other molecule in nature. For example, an exogenous region of a
construct could include a coding sequence flanked by sequences not
found in association with the coding sequence in nature. Another
example of an exogenous coding sequence is a construct where the
coding sequence itself is not found in nature (e.g., synthetic
sequences having codons different from the native gene). Similarly,
a host cell transformed with a construct which is not normally
present in the host cell would be considered exogenous for purposes
of this invention.
[0074] "Exogenous protein" as used herein refers to a protein not
naturally present in a particular tissue or cell, a protein that is
the expression product of an exogenous expression construct or
transgene, or a protein not naturally present in a given quantity
in a particular tissue or cell.
[0075] "Endogenous gene" refers to a naturally occurring gene or
fragment thereof normally associated with a particular cell.
[0076] The expression products described herein may consist of
proteinaceous material having a defined chemical structure.
However, the precise structure depends on a number of factors,
particularly chemical modifications common to proteins. For
example, since all proteins contain ionizable amino and carboxyl
groups, the protein may be obtained in acidic or basic salt form,
or in neutral form. The primary amino acid sequence may be
derivatized using sugar molecules (glycosylation) or by other
chemical derivatizations involving covalent or ionic attachment
with, for example, lipids, phosphate, acetyl groups and the like,
often occurring through association with saccharides. These
modifications may occur in vitro, or in vivo, the latter being
performed by a host cell through posttranslational processing
systems. Such modifications may increase or decrease the biological
activity of the molecule, and such chemically modified molecules
are also intended to come within the scope of the invention.
[0077] Alternative methods of cloning, amplification, expression,
and purification will be apparent to the skilled artisan.
Representative methods are disclosed in Sambrook, Fritsch, and
Maniatis, Molecular Cloning, a Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory (1989).
[0078] "Vector" means a polynucleotide comprised of single strand,
double strand, circular, or supercoiled DNA or RNA. A typical
vector may be comprised of the following elements operatively
linked at appropriate distances for allowing functional gene
expression: replication origin, promoter, enhancer, 5' mRNA leader
sequence, ribosomal binding site, nucleic acid cassette,
termination and polyadenylation sites, and selectable marker
sequences. One or more of these elements may be omitted in specific
applications. The nucleic acid cassette can include a restriction
site for insertion of the nucleic acid sequence to be expressed. In
a functional vector the nucleic acid cassette contains the nucleic
acid sequence to be expressed including translation initiation and
termination sites. An intron optionally may be included in the
construct, preferably .gtoreq.100 bp 5' to the coding sequence. A
vector is constructed so that the particular coding sequence is
located in the vector with the appropriate regulatory sequences,
the positioning and orientation of the coding sequence with respect
to the control sequences being such that the coding sequence is
transcribed under the "control" of the control or regulatory
sequences. Modification of the sequences encoding the particular
protein of interest may be desirable to achieve this end. For
example, in some cases it may be necessary to modify the sequence
so that it may be attached to the control sequences with the
appropriate orientation; or to maintain the reading frame. The
control sequences and other regulatory sequences may be ligated to
the coding sequence prior to insertion into a vector.
Alternatively, the coding sequence can be cloned directly into an
expression vector which already contains the control sequences and
an appropriate restriction site which is in reading frame with and
under regulatory control of the control sequences.
[0079] A "promoter" is a site on the DNA to which RNA polymerase
binds to initiate transcription of a gene. In some embodiments the
promoter will be modified by the addition or deletion of sequences,
or replaced with alternative sequences, including natural and
synthetic sequences as well as sequences which may be a combination
of synthetic and natural sequences. Many eukaryotic promoters
contain two types of recognition sequences: the TATA box and the
upstream promoter elements. The former, located upstream of the
transcription initiation site, is involved in directing RNA
polymerase to initiate transcription at the correct site, while the
latter appears to determine the rate of transcription and is
upstream of the TATA box. Enhancer elements can also stimulate
transcription from linked promoters, but many function exclusively
in a particular cell type. Many enhancer/promoter elements derived
from viruses, e.g., the SV40 promoter, the cytomegalovirus (CMV)
promoter, the rous-sarcoma virus (RSV) promoter, and the murine
leukemia virus (MLV) promoter are all active in a wide array of
cell types, and are termed "constitutive" or "ubiquitous".
Alternatively, non-constitutive promoters such as the mouse mammary
tumor virus (MMTV) promoter may also be used in the instant
invention. The nucleic acid sequence inserted in the cloning site
may have any open reading frame encoding a polypeptide of interest,
with the proviso that where the coding sequence encodes a
polypeptide of interest, it should lack cryptic splice sites which
can block production of appropriate mRNA molecules and/or produce
aberrantly spliced or abnormal mRNA molecules.
[0080] A "marker gene" is a gene which encodes a protein that
allows for identification and isolation of correctly transfected
cells. Suitable marker sequences include, but are not limited to
green, yellow, and blue fluorescent protein genes (GFP, YFP, and
BFP, respectively). Other suitable markers include thymidine kinase
(tk), dihydrofolate reductase (DHFR), and aminoglycoside
phosphotransferase (APH) genes. The latter imparts resistance to
the aminoglycoside antibiotics, such as kanamycin, neomycin, and
geneticin. These, and other marker genes such as those encoding
chloramphenicol acetyltransferase (CAT), .beta.-lactamase,
.beta.-galactosidase (.beta.-gal), may be incorporated into the
primary nucleic acid cassette along with the gene expressing the
desired protein, or the selection markers may be contained on
separate vectors and cotransfected.
[0081] A "reporter gene" is a marker gene that "reports" its
activity in a cell by the presence of the protein that it
encodes.
[0082] A "retroviral particle", "transducing particle", or
"transduction particle" refers to a replication-defective or
replication-competent virus capable of transducing non-viral DNA or
RNA into a cell.
[0083] The terms "transformation", "transduction" and
"transfection" all denote the introduction of a polynucleotide into
an avian blastodermal cell.
[0084] "Magnum" is that part of the oviduct between the
infundibulum and the isthmus containing tubular gland cells that
synthesize and secrete the egg white proteins of the egg.
[0085] A "MDOT promoter", as used herein, is a synthetic promoter
which is active in the tubular gland cells of the magnum of the
oviduct amongst other tissues. MDOT is comprised of elements from
the ovomucoid (MD) and ovotransferrin (TO) promoters (FIG. 13).
[0086] The term "optimized" is used in the context of "optimized
coding sequence", wherein the most frequently used codons for each
particular amino acid found in the egg white proteins ovalbumin,
lysozyme, ovomucoid, and ovotransferrin are used in the design of
the optimized human interferon-.alpha. 2b (IFN-.alpha. 2b)
polynucleotide sequence that is inserted into vectors of the
instant invention. More specifically, the DNA sequence for
optimized human IFN-.alpha. 2b is based on the hen oviduct
optimized codon usage and is created using the BACKTRANSLATE
program of the Wisconsin Package, Version 9.1 (Genetics Computer
Group Inc., Madison, Wis.) with a codon usage table compiled from
the chicken (Gallus gallus) ovalbumin, lysozme, ovomucoid, and
ovotransferrin proteins. For example, the percent usage for the
four codons of the amino acid alanine in the four egg white
proteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG.
Therefore, GCU is used as the codon for the majority of alanines in
the optimized human IFN-.alpha. 2b coding sequence. The vectors
containing the gene for optimized human IFN-.alpha. 2b are used to
create transgenic avians that express transgenic poultry derived
IFN-.alpha. 2b (TPD IFN-.alpha. 2b) in their tissues and eggs.
Similarly, the above method is employed for the design of the
optimized human erythropoietin (EPO) polynucleotide sequence in
order to create transgenic avians that express transgenic poultry
derived erythropoietin (TPD EPO) in their tissues and eggs.
[0087] b) Novel Vectors and Transgenesis of Blastodermal Cells
[0088] By the methods of the present invention, transgenes can be
introduced into avian embryonic blastodermal cells, to produce a
transgenic chicken or transgenic turkey, or other avian species,
that carries the transgene in the genetic material of its germ-line
tissue. The blastodermal cells are typically stage VII-XII cells,
or the equivalent thereof, and preferably are near stage X. The
cells useful in the present invention include embryonic germ (EG)
cells, embryonic stem (ES) cells & primordial germ cells
(PGCs). The embryonic blastodermal cells may be isolated freshly,
maintained in culture, or reside within an embryo.
[0089] The vectors useful in carrying out the methods of the
present invention are described herein. These vectors may be used
for stable introduction of an exogenous coding sequence into the
genome of an avian. Alternatively, the vectors may be used to
produce exogenous proteins in specific tissues of an avian, and in
the oviduct in particular. The vectors may also be used in methods
to produce avian eggs which contain exogenous protein. In a
preferred embodiment, the vector is retroviral and the coding
sequence and the promoter are both positioned between the 5' and 3'
LTRs of the retroviral vector. In another preferred embodiment, the
retroviral vector is derived from the avian leukosis virus (ALV),
murine leukemia virus (MLV), or lentivirus. In another preferred
embodiment, the vector includes a signal peptide coding sequence
which is operably linked to the coding sequence, so that upon
translation in a cell, the signal peptide will direct secretion of
the exogenous protein expressed by the vector into the egg white of
a hard shell egg. In yet another preferred embodiment, the vector
further includes a marker gene, wherein said marker gene is
operably linked to the promoter.
[0090] In some cases, introduction of a vector of the present
invention into the embryonic blastodermal cells is performed with
embryonic blastodermal cells that are either freshly isolated or in
culture. The transgenic cells are then typically injected into the
subgerminal cavity beneath a recipient blastoderm in an egg. In
some cases, however, the vector is delivered directly to the cells
of a blastodermal embryo.
[0091] In one embodiment of the invention, vectors used for
transfecting blastodermal cells and generating random, stable
integration into the avian genome contain a coding sequence and a
promoter in operational and positional relationship to express the
coding sequence in the tubular gland cell of the magnum of the
avian oviduct, wherein the coding sequence codes for an exogenous
protein which is deposited in the egg white of a hard shell egg.
The promoter may optionally be a segment of the ovalbumin promoter
region which is sufficiently large to direct expression of the
coding sequence in the tubular gland cells. The invention involves
truncating the ovalbumin promoter and/or condensing the critical
regulatory elements of the ovalbumin promoter so that it retains
sequences required for expression in the tubular gland cells of the
magnum of the oviduct, while being small enough that it can be
readily incorporated into vectors. In a preferred embodiment, a
segment of the ovalbumin promoter region may be used. This segment
comprises the 5'-flanking region of the ovalbumin gene. The total
length of the ovalbumin promoter segment may be from about 0.88 kb
to about 7.4 kb in length, and is preferably from about 0.88 kb to
about 1.4 kb in length. The segment preferably includes both the
steroid-dependent regulatory element and the negative regulatory
element of the ovalbumin gene. The segment optionally also includes
residues from the 5' untranslated region (5' UTR) of the ovalbumin
gene. Hence, the promoter may be derived from the promoter regions
of the ovalbumin-, lysozyme-, conalbumin-, ovomucoid-,
ovotransferrin- or ovomucin genes (FIG. 14). An example of such a
promoter is the synthetic MDOT promoter which is comprised of
elements from the ovomucoid and ovotransferrin promoter (FIG. 13).
The promoter may also be a promoter that is largely, but not
entirely, specific to the magnum, such as the lysozyme promoter.
The promoter may also be a mouse mammary tumor virus (MMTV)
promoter. Alternatively, the promoter may be a constitutive
promoter (e.g., a cytomegalovirus (CMV) promoter, a rous-sarcoma
virus (RSV) promoter, a murine leukemia virus (MLV) promoter,
etc.). In a preferred embodiment of the invention, the promoter is
a cytomegalovirus (CMV) promoter, a MDOT promoter, a rous-sarcoma
virus (RSV) promoter, a murine leukemia virus (MLV) promoter, a
mouse mammary tumor virus (MMTV) promoter, an ovalbumin promoter, a
lysozyme promoter, a conalbumin promoter, an ovomucoid promoter, an
ovomucin promoter, and an ovotransferrin promoter. Optionally, the
promoter may be at least one segment of a promoter region, such as
a segment of the ovalbumin-, lysozyme-, conalbumin-, ovomucoid-,
ovomucin-, and ovotransferrin promoter region. In a particularly
preferred embodiment, the promoter is a CMV promoter.
[0092] FIGS. 1A and 1B illustrate examples of ovalbumin promoter
expression vectors. Gene X is a coding sequence which encodes an
exogenous protein. Bent arrows indicate the transcriptional start
sites. In one example, the vector contains 1.4 kb of the 5'
flanking region of the ovalbumin gene (FIG. 1A). The sequence of
the "-1.4 kb promoter" of FIG. 1A corresponds to the sequence
starting from approximately 1.4 kb upstream (1.4 kb) of the
ovalbumin transcription start site and extending approximately 9
residues into the 5' untranslated region of the ovalbumin gene. The
approximately 1.4 kb-long segment harbors two critical regulatory
elements, the steroid-dependent regulatory element (SDRE) and the
negative regulatory element (NRE). The NRE is so named because it
contains several negative regulatory elements which block the
gene's expression in the absence of hormones (e.g., estrogen). A
shorter 0.88 kb segment also contains both elements. In another
example, the vector contains approximately 7.4 kb of the 5'
flanking region of the ovalbumin gene and harbors two additional
elements (HS-III and HS-IV), one of which is known to contain a
functional region enabling induction of the gene by estrogen (FIG.
1B). A shorter 6 kb segment also contains all four elements and
could optionally be used in the present invention.
[0093] Each vector used for random integration according to the
present invention preferably comprises at least one 1.2 kb element
from the chicken .beta.-globin locus which insulates the gene
within from both activation and inactivation at the site of
insertion into the genome. In a preferred embodiment, two insulator
elements are added to one end of the ovalbumin gene construct. In
the .beta.-globin locus, the insulator elements serve to prevent
the distal locus control region (LCR) from activating genes
upstream from the globin gene domain, and have been shown to
overcome position effects in transgenic flies, indicating that they
can protect against both positive and negative effects at the
insertion site. The insulator element(s) are only needed at either
the 5' or 3' end of the gene because the transgenes are integrated
in multiple, tandem copies effectively creating a series of genes
flanked by the insulator of the neighboring transgene. In another
embodiment, the insulator element is not linked to the vector but
is cotransfected with the vector. In this case, the vector and the
element are joined in tandem in the cell by the process of random
integration into the genome.
[0094] Each vector may optionally also comprise a marker gene to
allow identification and enrichment of cell clones which have
stably integrated the expression vector. The expression of the
marker gene is driven by a ubiquitous promoter that drives high
levels of expression in a variety of cell types. In a preferred
embodiment of the invention, the marker gene is human interferon
driven by a lysozyme promoter. In another embodiment the green
fluorescent protein (GFP) reporter gene (Zolotukhin et al., J.
Virol 70:4646-4654 (1995)) is driven by the Xenopus elongation
factor 1-.alpha. (ef-1.alpha.) promoter (Johnson and Krieg, Gene
147:223-26 (1994)). The Xenopus ef-1.alpha. promoter is a strong
promoter expressed in a variety of cell types. The GFP contains
mutations that enhance its fluorescence and is humanized, or
modified such that the codons match the codon usage profile of
human genes. Since avian codon usage is virtually the same as human
codon usage, the humanized form of the gene is also highly
expressed in avian blastodermal cells. In alternative embodiments,
the marker gene is operably linked to one of the ubiquitous
promoters of HSV tk, CMV, .beta.-actin, or RSV.
[0095] While human and avian codon usage is well matched, where a
nonvertebrate gene is used as the coding sequence in the transgene,
the nonvertebrate gene sequence may be modified to change the
appropriate codons such that codon usage is similar to that of
humans and avians.
[0096] Transfection of the blastodermal cells may be mediated by
any number of methods known to those of ordinary skill in the art.
The introduction of the vector to the cell may be aided by first
mixing the nucleic acid with polylysine or cationic lipids which
help facilitate passage across the cell membrane. However,
introduction of the vector into a cell is preferably achieved
through the use of a delivery vehicle such as a liposome or a
virus. Viruses which may be used to introduce the vectors of the
present invention into a blastodermal cell include, but are not
limited to, retroviruses, adenoviruses, adeno-associated viruses,
herpes simplex viruses, and vaccinia viruses.
[0097] In one method of transfecting blastodermal cells, a packaged
retroviral-based vector is used to deliver the vector into
embryonic blastodermal cells so that the vector is integrated into
the avian genome.
[0098] As an alternative to delivering retroviral transduction
particles to the embryonic blastodermal cells in an embryo, helper
cells which produce the retrovirus can be delivered to the
blastoderm.
[0099] A preferred retrovirus for randomly introducing a transgene
into the avian genome is the replication-deficient avian leucosis
virus (ALV), the replication-deficient murine leukemia virus (MLV),
or the lentivirus. In order to produce an appropriate retroviral
vector, a pNLB vector is modified by inserting a region of the
ovalbumin promoter and one or more exogenous genes between the 5'
and 3' long terminal repeats (LTRs) of the retrovirus genome. Any
coding sequence placed downstream of the ovalbumin promoter will be
expressed in the tubular gland cells of the oviduct magnum because
the ovalbumin promoter drives the expression of the ovalbumin
protein and is active in the oviduct tubular gland cells. While a
7.4 kb ovalbumin promoter has been found to produce the most active
construct when assayed in cultured oviduct tubular gland cells, the
ovalbumin promoter is preferably shortened for use in the
retroviral vector. In a preferred embodiment, the retroviral vector
comprises a 1.4 kb segment of the ovalbumin promoter; a 0.88 kb
segment would also suffice.
[0100] Any of the vectors of the present invention may also
optionally include a coding sequence encoding a signal peptide that
will direct secretion of the protein expressed by the vector's
coding sequence from the tubular gland cells of the oviduct. This
aspect of the invention effectively broadens the spectrum of
exogenous proteins that may be deposited in avian eggs using the
methods of the invention. Where an exogenous protein would not
otherwise be secreted, the vector bearing the coding sequence is
modified to comprise a DNA sequence comprising about 60 bp encoding
a signal peptide from the lysozyme gene. The DNA sequence encoding
the signal peptide is inserted in the vector such that it is
located at the N-terminus of the protein encoded by the cDNA.
[0101] FIGS. 2A-2D illustrate examples of suitable retroviral
vector constructs. The vector construct is inserted into the avian
genome with 5' and 3' flanking LTRs. Neo is the neomycin
phosphotransferase gene. Bent arrows indicate transcription start
sites. FIGS. 2A and 2B illustrate LTR and oviduct transcripts with
a sequence encoding the lysozyme signal peptide (LSP), whereas
FIGS. 2C and 2D illustrate transcripts without such a sequence.
There are two parts to the retroviral vector strategy. Any protein
that contains a eukaryotic signal peptide may be cloned into the
vectors depicted in FIGS. 2B and 2D. Any protein that is not
ordinarily secreted may be cloned into the vectors illustrated in
FIGS. 2A and 2B to enable its secretion from the tubular gland
cells.
[0102] FIG. 2E illustrates the strategy for cloning an exogenous
gene into a lysozyme signal peptide vector. The polymerase chain
reaction is used to amplify a copy of a coding sequence, gene X,
using a pair of oligonucleotide primers containing restriction
enzyme sites that enable the insertion of the amplified gene into
the plasmid after digestion with the two enzymes. The 5' and 3'
oligonucleotides contain the Bsu36I and Xba1 restriction sites,
respectively.
[0103] Another aspect of the invention involves the use of internal
ribosome entry site (IRES) elements in any of the vectors of the
present invention to allow the translation of two or more proteins
from a di- or polycistronic mRNA (Example 15). The IRES units are
fused to 5' ends of one or more additional coding sequences which
are then inserted into the vectors at the end of the original
coding sequence, so that the coding sequences are separated from
one another by an IRES (FIGS. 2F, 15A and 15D). Pursuant to this
aspect of the invention, post-translational modification of the
product is facilitated because one coding sequence may encode an
enzyme capable of modifying the other coding sequence product. For
example, the first coding sequence may encode collagen which would
be hydroxylated and made active by the enzyme encoded by the second
coding sequence. In the retroviral vector example of FIG. 2F, an
internal ribosome entry site (IRES) element is positioned between
two exogenous coding sequences (gene X and gene Y). The IRES allows
both protein X and protein Y to be translated from the same
transcript directed by an ovalbumin promoter. Bent arrows indicate
transcription start sites. The expression of the protein encoded by
gene X is expected to be highest in tubular gland cells, where it
is specifically expressed but not secreted. The protein encoded by
gene Y is also expressed specifically in tubular gland cells but
because it is efficiently secreted, protein Y is packaged into the
eggs. In the retroviral vector example of FIGS. 15A and 15D, the
light chain (LC) and heavy chain (HC) of a human monoclonal
antibody are expressed from a single vector, pCMV-LC-emcvIRES-HC,
by placement of an IRES from the encephalomyocarditis virus (EMCV).
Transcription is driven by a CMV promoter. (See also Murakami et
al. (1997) "High-level expression of exogenous genes by
replication-competent retrovirus vectors with an internal ribosomal
entry site" Gene 202:23-29; Chen et al. (1999) "Production and
design of more effective avian replication-incompetent retroviral
vectors" Dev. Biol. 214:370-384; Noel et al. (2000) "Sustained
systemic delivery of monoclonal antibodies by genetically modified
skin fibroblasts" J. Invest. Dermatol. 115:740-745.)
[0104] In another aspect of the invention, the coding sequences of
vectors used in any of the methods of the present invention are
provided with a 3' untranslated region (3' UTR) to confer stability
to the RNA produced. When a 3' UTR is added to a retroviral vector,
the orientation of the fused ovalbumin promoter, gene X and the 3'
UTR must be reversed in the construct, so that the addition of the
3' UTR will not interfere with transcription of the full-length
genomic RNA. In a preferred embodiment, the 3' UTR may be that of
the ovalbumin or lysozyme genes, or any 3' UTR that is functional
in a magnum cell, i.e., the SV40 late region.
[0105] In an alternative embodiment of the invention, a
constitutive promoter (e.g., CMV) is used to express the coding
sequence of a transgene in the magnum of an avian. In this case,
expression is not limited to the magnum; expression also occurs in
other tissues within the avian (e.g., blood). The use of such a
transgene, which includes a constitutive promoter and a coding
sequence, is particularly suitable for effecting the expression of
a protein in the oviduct and the subsequent secretion of the
protein into the egg white (see FIG. 8A for an example of a CMV
driven construct, such as the pNLB-CMV-IFN vector for expressing
IFN-.alpha. 2b in chickens).
[0106] FIG. 3A shows a schematic of the replication-deficient avian
leukosis virus (ALV)-based vector pNLB, a vector which is suitable
for use in this embodiment of the invention. In the pNLB vector,
most of the ALV genome is replaced by the neomycin resistance gene
(Neo) and the lacZ gene, which encodes b-galactosidase. FIG. 3B
shows the vector pNLB-CMV-BL, in which lacZ has been replaced by
the CMV promoter and the .beta.-lactamase coding sequence
(.beta.-La or BL). Construction of the vector is reported in the
specific examples (Example 1, vide infra). .beta.-lactamase is
expressed from the CMV promoter and utilizes a polyadenylation
signal (pA) in the 3' long terminal repeat (LTR). The
.beta.-Lactamase protein has a natural signal peptide; thus, it is
found in blood and in egg white.
[0107] Avian embryos are transduced with the pNLB-CMV-BL vector
(Example 2, vide infra). The egg whites of eggs from the resulting
stably transduced hens contain up to 60 micrograms (.mu.g) of
secreted, active .beta.-lactamase per egg (Examples 2 and 3, vide
infra).
[0108] FIGS. 8A and 8B illustrates the pNLB-CMV-IFN vector used for
expressing interferon-.alpha. 2b (IFN-.alpha. 2b) and the
pNLB-MDOT-EPO vector used for expressing erythropoietin (EPO),
respectively. Both exogenous proteins (EPO, IFN) are expressed in
avians, preferably chicken and turkey.
[0109] The pNLB-MDOT-EPO vector is created by substituting an EPO
encoding sequence for the BL encoding sequence (Example 10, vide
infra). In one embodiment, a synthetic promoter called MDOT is
employed to drive expression of EPO. MDOT contains elements from
both the ovomucoid and ovotransferrin promoter. The DNA sequence
for human EPO is based on hen oviduct optimized codon usage as
created using the BACKTRANSLATE program of the Wisconsin Package,
version 9.1 (Genetics Computer Group, Inc., Madison, Wis.) with a
codon usage table compiled from the chicken (Gallus gallus)
ovalbumin, lysozyme, ovomucoid, and ovotransferrin proteins. The
EPO DNA sequence is synthesized and cloned into the vector and the
resulting plasmid is pNLB-MDOT-EPO (a.k.a. pAVIJCR-A145.27.2.2). In
one embodiment, transducing particles (i.e., transduction
particles) are produced for the vector, and these transducing
particles are titered to determine the appropriate concentration
that can be used to inject embryos. Eggs are then injected with
transducing particles after which they hatch about 21 days later.
The exogenous protein levels such as the EPO levels can then be
measured by an ELISA assay from serum samples collected from chicks
one week after hatch. Male birds are selected for breeding, wherein
birds are screened for G.sub.0 roosters which contain the EPO
transgene in their sperm. Preferably, roosters with the highest
levels of the transgene in their sperm samples are bred to
nontransgenic hens by artificial insemination. Blood DNA samples
are screened for the presence of the transgene. A number of chicks
are usually found to be transgenic (G.sub.1 avians). Chick serum is
tested for the presence of human EPO (e.g., ELISA assay). The egg
white in eggs from G.sub.1 hens is also tested for the presence of
human EPO. The EPO (i.e., derived from the optimized coding
sequence of human EPO) present in eggs of the instant invention is
biologically active (Example 11).
[0110] Similarly, the pNLB-CMV-IFN vector (FIG. 8A) is created by
substituting an IFN encoding sequence for the BL encoding sequence
(Example 12, vide infra). In one embodiment, a constitutive
cytomegalovirus (CMV) promoter is employed to drive expression of
IFN. More specifically, the IFN coding sequence is controlled by
the cytomegalovirus (CMV) immediate early promoter/enhancer and
SV40 polyA site. FIG. 8A illustrates pNLB-CMV-IFN used for
expressing IFN in avians, preferably chicken and turkey. An
optimized coding sequence is created for human IFN-.alpha. 2b,
wherein the most frequently used codons for each particular amino
acid found in the egg white proteins ovalbumin, lysozyme,
ovomucoid, and ovotransferrin are used in the design of the human
IFN-.alpha. 2b sequence that is inserted into vectors of the
instant invention. More specifically, the DNA sequence for the
optimized human IFN-.alpha. 2b (FIG. 11A) is based on the hen
oviduct optimized codon usage and is created using the
BACKTRANSLATE program (supra) with a codon usage table compiled
from the chicken (Gallus gallus) ovalbumin, lysozme, ovomucoid, and
ovotransferrin proteins. For example, the percent usage for the
four codons of the amino acid alanine in the four egg white
proteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG.
Therefore, GCU is used as the codon for the majority of alanines in
the optimized human IFN-.alpha. 2b sequence. The vectors containing
the gene for the optimized human IFN-.alpha. 2b sequence are used
to create transgenic avians that express TPD IFN-.alpha. 2b in
their tissues and eggs.
[0111] Transducing particles (i.e., transduction particles) are
produced for the vector and titered to determine the appropriate
concentration that can be used to inject embryos (Example 2, vide
infra). Thus, chimeric avians are produced (see also Example 13,
vide infra). Avian eggs are windowed according to the Speksnijder
procedure (U.S. Pat. No. 5,897,998), and eggs are injected with
transducing particles Eggs hatch about 21 days after injection.
hIFN levels are measured (e.g., ELISA assay) from serum samples
collected from chicks one week after hatch. As with EPO (supra),
male birds are selected for breeding. In order to screen for Go
roosters which contain the IFN transgene in their sperm, DNA is
extracted from rooster sperm samples. The G.sub.0 roosters with the
highest levels of the transgene in their sperm samples are bred to
nontransgenic hens by artificial insemination. Blood DNA samples
are screened for the presence of the transgene. The serum of
transgenic roosters is tested for the presence of hIFN (e.g., ELISA
assay). If the exogenous protein is confirmed the sperm of the
transgenic roosters is used for artificial insemination of
nontransgenic hens. A certain percent of the offspring will then
contain the transgene (e.g., more than 50%). When IFN (i.e.,
derived from the optimized coding sequence of human IFN) is present
in eggs of the instant invention, the IFN may be tested for
biological activity. As with EPO, such eggs usually contain
biologically active IFN, such as TPD IFN-.alpha. 2b (FIG. 11B).
[0112] c) Production of Transgenic Avians and Exogenous Proteins in
Eggs
[0113] The methods of the invention which provide for the
production of exogenous protein in the avian oviduct and the
production of eggs which contain exogenous protein involve an
additional step subsequent to providing a suitable vector and
introducing the vector into embryonic blastodermal cells so that
the vector is integrated into the avian genome. The subsequent step
involves deriving a mature transgenic avian from the transgenic
blastodermal cells produced in the previous steps. Deriving a
mature transgenic avian from the blastodermal cells optionally
involves transferring the transgenic blastodermal cells to an
embryo and allowing that embryo to develop fully, so that the cells
become incorporated into the avian as the embryo is allowed to
develop. The resulting chick is then grown to maturity. In a
preferred embodiment, the cells of a blastodermal embryo are
transfected or transduced with the vector directly within the
embryo (Example 2). The resulting embryo is allowed to develop and
the chick allowed to mature.
[0114] In either case, the transgenic avian so produced from the
transgenic blastodermal cells is known as a founder. Some founders
will carry the transgene in the tubular gland cells in the magnum
of their oviducts. These avians will express the exogenous protein
encoded by the transgene in their oviducts. The exogenous protein
may also be expressed in other tissues (e.g., blood) in addition to
the oviduct. If the exogenous protein contains the appropriate
signal sequence(s), it will be secreted into the lumen of the
oviduct and into the egg white of the egg. Some founders are
germ-line founders (Examples 8 and 9). A germ-line founder is a
founder that carries the transgene in genetic material of its
germ-line tissue, and may also carry the transgene in oviduct
magnum tubular gland cells that express the exogenous protein.
Therefore, in accordance with the invention, the transgenic avian
will have tubular gland cells expressing the exogenous protein, and
the offspring of the transgenic avian will also have oviduct magnum
tubular gland cells that express the exogenous protein.
Alternatively, the offspring express a phenotype determined by
expression of the exogenous gene in specific tissue(s) of the avian
(Example 6, Table 2). In a preferred embodiment of the invention,
the transgenic avian is a chicken or a turkey.
[0115] The invention can be used to express, in large yields and at
low cost, a wide range of desired proteins including those used as
human and animal pharmaceuticals, diagnostics, and livestock feed
additives. Proteins such as interferon (IFN), erythropoietin (EPO),
human growth hormone, lysozyme, and .beta.-casein are examples of
proteins which are desirably expressed in the oviduct and deposited
in eggs according to the invention (Examples 2, 3, and 5). Other
possible proteins to be produced include, but are not limited to,
albumin, .alpha.-1 antitrypsin, antithrombin III, collagen, factors
VIII, IX, X (and the like), fibrinogen, hyaluronic acid, insulin,
lactoferrin, protein C, granulocyte colony-stimulating factor
(G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF),
tissue-type plasminogen activator (tPA), feed additive enzymes,
somatotropin, and chymotrypsin. Genetically engineered antibodies,
such as immunotoxins which bind to surface antigens on human tumor
cells and destroy them, can also be expressed for use as
pharmaceuticals or diagnostics.
[0116] d) Transgenic Poultry Derived Interferon-.alpha. 2b (TPD
IFN-.alpha. 2b)
[0117] The instant invention encompasses a transgenic poultry
derived interferon-.alpha. 2b (TPD IFN-.alpha. 2b) derived from
avians. TPD IFN-.alpha. 2b exhibits a new glycosylation pattern and
contains two new glyco forms (bands 4 and 5 are .alpha.-Gal
extended disaccharides; see FIG. 9) not normally seen in human
peripheral blood leukocyte derived interferon-.alpha. 2b (PBL
IFN-.alpha. 2b). TPD IFN-.alpha. 2b also contains O-linked
carbohydrate structures that are similar to human PBL IFN-.alpha.
2b and is more efficiently produced in chickens then the human
form.
[0118] The instant invention contemplates an isolated
polynucleotide comprising the optimized polynucleotide sequence of
human IFN-.alpha. 2b, i.e., recombinant transgenic poultry derived
interferon-.alpha. 2b (TPD IFN-.alpha. 2b) coding sequence (SEQ ID
NO: 1). The coding sequence for optimized human IFN-.alpha. 2b
includes 498 nucleic acids and 165 amino acids (see SEQ ID NO: 1
and FIG. 11A). Similarly, the coding sequence for natural human
IFN-.alpha. 2b includes 498 nucleic acids (NCBI Accession Number
AF405539 and GI:15487989) and 165 amino acids (NCBI Accession
Number AAL01040 and GI:15487990). The most frequently used codons
for each particular amino acid found in the egg white proteins
ovalbumin, lysozyme, ovomucoid, and ovotransferrin are used in the
design of the optimized human IFN-.alpha. 2b coding sequence which
is inserted into vectors of the instant invention. More
specifically, the DNA sequence for the optimized human IFN-.alpha.
2b is based on the hen oviduct optimized codon usage and is created
using the BACKTRANSLATE program of the Wisconsin Package, Version
9.1 (Genetics Computer Group Inc., Madison, Wis.) with a codon
usage table compiled from the chicken (Gallus gallus) ovalbumin,
lysozme, ovomucoid, and ovotransferrin proteins. For example, the
percent usage for the four codons of the amino acid alanine in the
four egg white proteins is 34% for GCU, 31% for GCC, 26% for GCA,
and 8% for GCG. Therefore, GCU is used as the codon for the
majority of alanines in the optimized human IFN-.alpha. 2b coding
sequence. The vectors containing the gene for optimized human
IFN-.alpha. 2b are used to create transgenic avians that express
TPD IFN-.alpha. 2b in their tissues and eggs.
[0119] As discussed in Example 13 (vide infra), TPD IFN-.alpha. 2b
is produced in chicken. However, TPD IFN-.alpha. 2b may also be
produced in turkey and other avian species. In a preferred
embodiment of the invention, TPD IFN-.alpha. 2b is expressed in
chicken and turkey and their hard shell eggs. A carbohydrate
analysis (Example 14, vide infra), including a monosaccharide
analysis and FACE analysis, reveals the sugar make-up or novel
glycosylation pattern of the protein. As such, TPD IFN-.alpha. 2b
shows the following monosaccharide residues: N-Acetyl-Galactosamine
(NAcGal), Galactose (Gal), N-Acetyl-Glucosamine (NAcGlu), and
Sialic acid (SA). However, there is no N-linked glycosylation in
TPD IFN-.alpha. 2b. Instead, TPD IFN-.alpha. 2b is O-glycosylated
at Thr-106. This type of glycosylation is similar to human
IFN-.alpha. 2, wherein the Thr residue at position 106 is unique to
IFN-.alpha. 2. Similar to natural IFN-.alpha., TPD IFN-.alpha. 2b
does not have mannose residues. A FACE analysis reveals 6 bands
(FIG. 9) that represent various sugar residues, wherein bands 1, 2
and 3 are un-sialyated, mono-sialyated, and di-sialyated,
respectively (FIG. 10). The sialic acid (SA) linkage is alpha 2-3
to Galactose (Gal) and alpha 2-6 to N-Acetyl-Galactosamine
(NAcGal). Band 6 represents an un-sialyated tetrasaccharide. Bands
4 and 5 are alpha-Galactose (alpha-Gal) extended disaccharides that
are not seen in human PBL IFN-.alpha. 2b or natural human IFN
(natural hIFN). FIG. 10 shows the comparison of TPD IFN-.alpha. 2b
(egg white hIFN) and human PBL IFN-.alpha. 2b (natural hIFN). Minor
bands are present between bands 3 and 4 and between bands 4 and 5
in TPD IFN-.alpha. 2b (vide infra).
[0120] The instant invention contemplates an isolated polypeptide
sequence (SEQ ID NO: 2) of TPD IFN-.alpha. 2b (see also FIG. 11B)
and a pharmaceutical composition thereof, wherein the protein is
O-glycosylated at Thr-106 with specific residues. These residues
are as follows: 1
[0121] wherein Gal=Galactose,
[0122] NAcGal=N-Acetyl-Galactosamine,
[0123] NAcGlu=N-Acetyl-Glucosamine, and
[0124] SA=Sialic Acid.
[0125] In a preferred embodiment of the instant invention, the
percentages are as follows: 2
[0126] Minor bands are present between bands 3 and 4 and between
bands 4 and 5 which account for about 17% in TPD IFN-.alpha.
2b.
e) EXAMPLES
[0127] The following specific examples are intended to illustrate
the invention and should not be construed as limiting the scope of
the claims.
Example 1
Vector Construction
[0128] The lacZ gene of pNLB, a replication-deficient avian
leukosis virus (ALV)-based vector (Cosset et al., 1991), was
replaced with an expression cassette consisting of a
cytomegalovirus (CMV) promoter and the reporter gene,
.beta.-lactamase. The pNLB and pNLB-CMV-BL vector constructs are
diagrammed in FIG. 3A and 3B, respectively.
[0129] To efficiently replace the lacZ gene of pNLB with a
transgene, an intermediate adaptor plasmid was first created,
pNLB-Adapter. pNLB-Adapter was created by inserting the chewed back
ApaI/ApaI fragment of pNLB (Cosset et al., J. Virol. 65:3388-94
(1991)) (in pNLB, the 5' ApaI resides 289 bp upstream of lacZ and
the 3'ApaI resides 3' of the 3' LTR and Gag segments) into the
chewed-back KpnI/SacI sites of pBluescriptKS(-). The filled-in
MluI/XbaI fragment of pCMV-BL (Moore et al., Anal. Biochem. 247:
203-9 (1997)) was inserted into the chewed-back KpnI/NdeI sites of
pNLB-Adapter, replacing lacZ with the CMV promoter and the BL gene
(in pNLB, KpnI resides 67 bp upstream of lacZ and NdeI resides 100
bp upstream of the lacZ stop codon), thereby creating
pNLB-Adapter-CMV-BL. To create pNLB-CMV-BL, the HindIII/BlpI insert
of pNLB (containing lacZ) was replaced with the HindIII/BlpI insert
of pNLB-Adapter-CMV-BL. This two step cloning was necessary because
direct ligation of blunt-ended fragments into the HindIII/BlpI
sites of pNLB yielded mostly rearranged subclones, for unknown
reasons.
Example 2
Creation of the NLB-CMV-BL Founder Flock
[0130] Sentas and Isoldes were cultured in F10 (Gibco), 5% newborn
calf serum (Gibco), 1% chicken serum (Gibco), 50 .mu.g/ml
phleomycin (Cayla Laboratories) and 50 .mu.g/ml hygromycin (Sigma).
Transduction particles were produced as described in Cosset et al.,
1993, herein incorporated by reference, with the following
exceptions. Two days after transfection of the retroviral vector
pNLB-CMV-BL (from Example 1, above) into 9.times.10.sup.5 Sentas,
virus was harvested in fresh media for 6-16 hours and filtered. All
of the media was used to transduce 3.times.10.sup.6 Isoldes in 3
100 mm plates with polybrene added to a final concentration of 4
.mu.g/ml. The following day the media was replaced with media
containing 50 .mu.g/ml phleomycin, 50 .mu.g/ml hygromycin and 200
.mu.g/ml G418 (Sigma). After 10-12 days, single G418.sup.r colonies
were isolated and transferred to 24-well plates. After 7-10 days,
titers from each colony were determined by transduction of Sentas
followed by G418 selection. Typically 2 out of 60 colonies gave
titers at 1-3.times.10.sup.5. Those colonies were expanded and
virus concentrated to 2-7.times.10.sup.6 as described in Allioli et
al., Dev. Biol. 165:30-7 (1994), herein incorporated by reference.
The integrity of the CMV-BL expression cassette was confirmed by
assaying for .beta.-lactamase in the media of cells transduced with
NLB-CMV-BL transduction particles.
[0131] The transduction vector, NLB-CMV-BL, was injected into the
subgerminal cavity of 546 unincubated SPF White Leghorn embryos, of
which 126 chicks hatched and were assayed for secretion of
.beta.-lactamase (lactamase) into blood. In order to measure the
concentration of active lactamase in unknown samples, a kinetic
colorimetric assay was employed in which PADAC, a purple substrate,
is converted to a yellow compound specifically by lactamase.
Lactamase activity was quantitated by monitoring the decrease in
OD.sub.570nm during a standard reaction time and compared to a
standard curve with varying levels of purified lactamase (referred
to as the "lactamase assay"). The presence or absence of lactamase
in a sample could also be determined by visually scoring for the
conversion of purple to yellow in a test sample overnight or for
several days (the "overnight lactamase assay"). The latter method
was suitable for detection of very low levels of lactamase or for
screening a large number of samples. At one to four weeks of age,
chick serum samples were tested for the presence of lactamase.
Twenty-seven chicks had very low levels of lactamase in their serum
that was detectable only after the overnight lactamase assay and,
as these birds matured, lactamase was no longer detectable. As
shown in Table 1 below and FIG. 4A, 9 additional birds (3 males and
6 females) had serum levels of lactamase that ranged from 11.9 to
173.4 ng/ml at six to seven months post-hatch.
1TABLE 1 Expression of .beta.-Lactamase in NLB-CMV-BL-Transduced
Chickens Average ng/ml of .beta.-Lactamase Egg White: Egg White:
Serum: 8 8 Month 14 Month Sex Band No. Month Birds Hens.sup.3
Hens.sup.3 NA.sup.1 Controls.sup.2 0.0 .+-. 7.4 0.0 .+-. 13.6 0.0
.+-. 8.0 Female 1522 36.7 .+-. 1.6 56.3 .+-. 17.8 47.9 .+-. 14.3
Female 1549 11.9 .+-. 1.3 187.0 .+-. 32.4 157.0 .+-. 32.2 Female
1581 31.5 .+-. 4.8 243.8 .+-. 35.7 321.7 .+-. 68.8 Female 1587 33.9
.+-. 1.4 222.6 .+-. 27.7 291.0 .+-. 27.0 Female 1790 31.0 .+-. 0.5
136.6 .+-. 20.2 136.3 .+-. 11.0 Female 1793 122.8 .+-. 3.6 250.0
.+-. 37.0 232.5 .+-. 28.6 Male 2395 16.0 .+-. 2.3 NA NA Male 2421
165.5 .+-. 5.0 NA NA Male 2428 173.4 .+-. 5.9 NA NA .sup.1NA: not
applicable. .sup.2Controls were obtained from untreated hens.
.sup.3Represents the average of 5 to 20 eggs.
Example 3
.beta.-Lactamase Expression in the Egg White of G0 Hens
[0132] Fifty-seven pullets transduced with NLB-CMV-BL retroviral
vector were raised to sexual maturity and egg white from each hen
was tested for active .beta.-lactamase (lactamase) at 8 months of
age. Of the 57 birds, six had significant levels of lactamase that
ranged from 56.3 to 250.0 ng/ml (Table 1, supra). No other hens in
this group had detectable levels of lactamase in their egg white,
even after incubation of PADAC with the sample for several days.
Lactamase was not detectable in egg white from 24 hens that were
mock injected and in 42 hens that were transduced with a NLB vector
that did not carry the lactamase transgene. Stable lactamase
expression was still detectable in the egg white of the six
expressing hens six months following the initial assays (Table 1,
supra).
[0133] Lactamase was detected in the egg white of all six hens by a
western blot assay with an anti-.beta.-lactamase antibody. The egg
white lactamase was the same size as the bacterially produced,
purified lactamase that was used as a standard. The amount detected
in egg white by Western analysis was consistent with that
determined by the enzymatic assay, indicating that a significant
proportion of the egg white lactamase was biologically active.
Hen-produced lactamase in egg white stored at 4.degree. C. lost no
activity and showed no change in molecular weight even after
several months of storage. This observation allowed storage of
lactamase-containing eggs for extended periods prior to
analysis.
Example 4
Germline Transmission and Serum Expression of the .beta.-Lactamase
Transgene in G1 and G2 Transgenic Chickens
[0134] DNA was extracted from sperm collected from 56 G0 roosters
and three of the 56 birds that harbored significant levels of the
transgene in their sperm DNA as determined by quantitative PCR were
selected for breeding. These roosters were the same three that had
the highest levels of .beta.-lactamase (lactamase) in their blood
(roosters 2395, 2421 and 2428). Rooster 2395 gave rise to three G1
transgenic offspring (out of 422 progeny) whereas the other two
yielded no transgenic offspring out of 630 total progeny. Southern
analysis of blood DNA from each of the three G1 transgenic chickens
confirmed that the transgenes were intact and that they were
integrated at unique random loci. The serum of the G1 transgenic
chicks, 5308, 5657 and 4133, at 6 to 11 weeks post-hatch contained
0.03, 2.0 and 6.0 .mu.g/ml of lactamase, respectively. The levels
of lactamase dropped to levels of 0.03, 1.1 and 5.0 .mu.g/ml when
the chickens were assayed again at 6 to 7 months of age (FIG.
4A).
[0135] Hen 5657 and rooster 4133 were bred to non-transgenic
chickens to obtain offspring hemizygous for the transgene. The
pedigrees of transgenic chickens bred from rooster 4133 or hen 5657
and the subsequent generations are shown in FIG. 5. Transgenic
rooster 5308 was also bred but this bird's progeny exhibited
lactamase concentrations that were either very low or not
detectable in serum and egg white. Active lactamase concentrations
in the serum of randomly selected G2 transgenic chicks were
measured at 3 to 90 days post-hatch. Of the five G2 transgenics
bred from hen 5657, all had active lactamase at concentrations of
1.9 to 2.3 .mu.g/ml (compared to the parental expression of 1.1
.mu.g/ml, FIG. 4B). All of the samples were collected during the
same period of time, thus, the lactamase concentrations in the
serum of the offspring were expected to be higher than that of the
parent since the concentration in hen 5657 had dropped
proportionately as she matured. Similarly, the five randomly
selected transgenic chicks bred from rooster 4133 all had serum
lactamase concentrations that were similar but higher than that of
their parent (FIG. 4B).
Example 5
.beta.-Lactamase Expression in the Egg White of Transgenic Hens
[0136] Eggs from G1 hen 5657 contained 130 ng of active
.beta.-lactamase (lactamase) per ml of egg white (FIG. 6A).
Lactamase concentrations were higher in the first few eggs laid and
then reached a plateau that was stable for at least nine months.
Eggs from transgenic hens bred from hen 5657 and a non-transgenic
rooster had lactamase concentrations that were similar to their
parent (FIG. 6A). Hen 6978 was bred from G2 hen 8617 and sibling G2
rooster 8839 and was homozygous for the transgene as determined by
quantitative PCR and Southern analysis. As expected, the
concentration of lactamase in the eggs of bird 6978 was nearly
two-fold higher than her hemizygous parent (FIG. 6B). No other G3
hens bred from hen 5657, were analyzed because hen 6978 was the
only female in her clutch. It is important to note that the eggs
from hens 8867, 8868 and 8869 were collected eleven months apart
and had similar concentrations of lactamase (FIGS. 6A and 6B),
again indicating that the expression levels in the egg white were
consistent throughout the lay period.
[0137] Rooster 4133 was bred to non-transgenic hens to obtain
hemizygous G2 hens. Of the 15 transgenic hens analyzed, all had
lactamase in the egg white at concentrations ranging from 0.47 to
1.34 .mu.g/ml. Four representative hens are shown in FIG. 7A. When
assayed 6 months later, the average expression level had dropped
from approximately 1.0 .mu.g/ml to 0.8 .mu.g/ml (FIG. 7A).
Expression levels were high in the initial eggs and leveled out
over several months. After that, the concentrations of lactamase in
the eggs remained constant.
[0138] G2 hen 8150 and sibling G2 rooster 8191 were crossed to
yield hemizygous and homozygous G3 hens. All transgenic G3 hens
expressed lactamase in the white of their eggs at concentrations
ranging from 0.52 to 1.65 .mu.g/ml (FIG. 7B). The average
expression for the G3 hens that were homozygous was 47% higher than
those G2 hens and G3 hens that were hemizygous. The amount of
lactamase in the eggs from G2 and G3 hens bred from rooster 4133
and his offspring varied significantly (FIGS. 7A and 7B), although
the levels in the eggs from any given hen in that group were
relatively constant. The average expression of lactamase was
expected to double for the homozygous genotype. Western blot
analysis confirmed that the transgene was faithfully producing
intact lactamase in the eggs of G2 transgenics. The lactamase level
detected on a Western blot also correlated closely with that
determined by the enzyme activity assay, indicating that a
significant portion of the egg white lactamase was bioactive. Thus,
retroviral vectors were successfully employed to implement stable
and reliable expression of a transgene in chickens.
[0139] Deposition of lactamase in the yolk was detectable but lower
than that of egg white. Seven G2 or G3 hens of rooster 4133's
lineage were analyzed and the concentration in the yolk ranged from
107 to 375 ng/ml or about 20% the concentration in the egg white.
There was no correlation between the yolk and egg white lactamase
levels of a given hen (Harvey et al., "Expression of exogenous
protein in egg white of transgenic chickens" (April 2002) Nat.
Biotechnol. 20:396-399).
Example 6
Production of Founder Males
[0140] For NLB-CMV-BL transduction, freshly laid fertilized White
Leghorn eggs were used. Seven to ten microliters of concentrated
particles were injected into the subgerminal cavity of windowed
eggs and chicks hatched after sealing the window. 546 eggs were
injected. Blood DNA was extracted and analyzed for the presence of
the transgene using a probe-primer set designed to detect the
neo.sup.r gene via the Taqman assay. As can be seen in Table 2
below, approximately 25% of all chicks had detectable levels of
transgene in their blood DNA.
2TABLE 2 Summary of Transgenesis with the NLB-CMV-BL Vectors
Transgene NLB-CMV-BL Number of injections 546 Number of birds
hatched (%) 126 (23.1%) Number of chicks with transgene in 36
(28.6%) their blood DNA (%) Number of males 56 Number of males with
transgene in 3 (5.4%) their sperm DNA (%) Production of G1 flock
Number of chicks bred from G0 males 1026 Number of G1 transgenics 3
Rate of germline transmission 0.29% Production of G2 flock Number
of chicks bred from G1 120 transgenics Number of G2 transgenics 61
Rate of germline transmission 50.8% Number of males that
transmitted 1 (1.8%) transgene to progeny (%)
Example 7
Germline Transmission of the Transgene
[0141] Taqman detection of the neo.sup.r gene in sperm DNA was used
to identify candidate G0 males for breeding. Three G0 males were
identified, wherein each had the NLB-CMV-BL transgene in their
sperm DNA at levels that were above background. All G0 males
positive for the transgene in their sperm were bred to
non-transgenic hens to identify fully transgenic G1 offspring.
[0142] For NLB-CMV-BL 1026 chicks were bred, respectively, and
three G1 chicks obtained for each transgene (Table 2, supra). All
G1 progeny came from the male with the highest level of transgene
in his sperm DNA, even though an equivalent number of chicks were
bred from each male.
Example 8
Southern Analysis of G1s and G2s
[0143] In order to confirm integration and integrity of the
inserted vector sequences, Southern blot analysis was performed on
DNA from G1 and G2 transgenics. Blood DNA was digested with HindIII
and hybridized to a neo.sup.r probe to detect junction fragments
created by the internal HindIII site found in the NLB-CMV-BL vector
(FIG. 3B) and genomic sites flanking the site of integration. Each
of the 3 G1 birds carrying NLB-CMV-BL had a junction fragment of
unique size, indicating that the transgene had integrated into
three different genomic sites. G1s were bred to non-transgenic hens
to obtain hemizygous G2s. As can be seen in Table 2 (supra), 50.8%
of offspring from G1 roosters harboring NLB-CMV-BL were transgenic
as expected for Mendelian segregation of a single integrated
transgene. Southern analysis of HindIII-digested DNA from G2
offspring detected junction fragments similar in size to those
originating from their transgenic parents, indicating that the
transgene was transmitted intact.
Example 9
Screening for G3 Progeny Homozygous for the Transgene
[0144] In order to obtain transgenic chickens homozygous for the
transgene, G2 hemizygous birds bearing NLB-CMV-BL integrated at the
same site (e.g., progeny of the same G1 male) were crossbred. Two
groups were bred: the first was a hen and rooster arising from the
G1 4133 male and the second from the G1 5657 hen. The Taqman assay
was used to quantitatively detect the neo.sup.r transgene in G3
progeny using a standard curve. The standard curve was constructed
using known amounts of genomic DNA from the G1 transgenic 4133 male
hemizygous for the transgene as determined by Southern analysis.
The standard curve ranged from 10.sup.3 to 1.6.times.10.sup.4 total
copies of the transgene or 0.2 to 3.1 transgene copies per diploid
genome. Because reaction components were not limited during the
exponential phase, amplification was very efficient and gave
reproducible values for a given copy number. There was a
reproducible, one-cycle difference between each standard curve
differing 2-fold in copy number.
[0145] In order to determine the number of transgene alleles in the
G3 offspring, DNAs were amplified and compared to the standards.
DNA from non-transgenics did not amplify. Birds homozygous for the
transgenic allele gave rise to plots initiating the amplification
one cycle earlier than those hemizygous for the allele. The
sequence detection program was able to calculate the number of
alleles in an unknown DNA sample based on the standard curve and
the cycle threshold (Ct) at which a sample's amplification plot
exhibited a significant rise. The data are shown in Table 3
below.
[0146] In order to confirm Taqman copy number analysis, DNA of
selected birds was analyzed by Southern blotting using
PstI-digested DNA and a probe complementary to the neo.sup.r gene
to detect a 0.9 kb fragment. Detection of a small fragment was
chosen since transfer of smaller DNAs from gel to membrane is more
quantitative. The signal intensity of the 0.9 kb band corresponded
well to the copy number of G3 transgenic birds as determined by the
Taqman assay. The copy numbers of an additional eighteen G3
transgenic birds analyzed by Southern blotting were also consistent
with that determined by Taqman. A total of 33 progeny were analyzed
for the 4133 lineage, of which 9 (27.3%) were non-transgenic, 16
(48.5%) were hemizygous and 8 (24.2%) were homozygous. A total of
10 progeny were analyzed for the 5657 lineage, of which 5 (50.0%)
were non-transgenic, 1 (10.0%) was hemizygous and 4 (40.0%) were
homozygous. The observed ratio of non-transgenics, hemizygotes and
homozygotes for the 4133 lineage G3 progeny was not statistically
different from the expected 1:2:1 ratio as determined by the
.chi..sup.2 test (P.ltoreq.0.05). Progeny of the 5657 lineage did
not have the expected distribution but this could have been due to
the low number of progeny tested (Harvey et al., "Consistent
production of transgenic chickens using replication deficient
retroviral vectors and high-throughput screening procedures"
(February 2002) Poultry Science 81:202-212).
3TABLE 3 Determination of Transgene Copy Number in G3 Offspring
Bred from G2 Transgenics Band No. Mean Total Copies per (Std. No.
Copy Standard Diploid G1 Parent or NTC.sup.1) Ct.sup.2 Number
Deviation Genome.sup.3 .sup. NA.sup.4 4133 27.3 3,975 145.7 1 4133
6792 40.0 0 0.0 0 5657 6977 25.9 10,510 587.0 2 5657 6978 25.8
10,401 505.1 2 4133 7020 26.7 6,064 443.1 1 4133 7021 26.8 5,239
133.8 1 4133 7022 26.1 9,096 352.3 2 4133 7023 26.8 5,424 55.7 1
4133 7024 26.9 4,820 110.1 1 5657 7110 26.4 8,092 1037.5 2 5657
7111 30.4 403 46.3 0 5657 7112 33.2 60 6.1 0 4133 7142 26.5 6,023
367.6 1 4133 7143 25.9 9,474 569.8 2 4133 7144 25.7 12,420 807.7 2
4133 7338 27.2 4,246 201.7 1 5657 7407 37.7 1 1.0 0 NA (std1) 29.1
1,000 0.0 0.2 NA (std2) 28.1 2,000 0.0 0.4 NA (std3) 27.1 4,000 0.0
0.8 NA (std4) 26.2 8,000 0.0 1.6 NA (std5) 25.3 16,000 0.0 3.1 NA
(NTC) 39.8 -1 0.0 0.0 .sup.1Std. No.: standard number; NTC: no
template control. .sup.2Ct: cycle threshold; cycle at which a
sample's fluorescence exhibited a significant increase above
background. .sup.3Copies per diploid genome were determined by
dividing the mean by 5100 and rounding to the nearest first decimal
place. .sup.4NA: not applicable.
Example 10
Vector Construction for pNLB-MDOT-EPO vector
[0147] Following the teachings of Example 1 (Vector Construction)
of the specification, an pNLB-MDOT-EPO vector was created,
substituting an EPO encoding sequence for the BL encoding sequence
(FIG. 8B). Instead of using the CMV promoter MDOT was used (FIG.
13). MDOT is a synthetic promoter which contains elements from both
the ovomucoid (MD) and ovotransferrin (TO) promoter. (pNLB-MDOT-EPO
vector, a.k.a. pAVIJCR-A145.27.2.2).
[0148] The DNA sequence for human EPO based on hen oviduct
optimized codon usage was created using the BACKTRANSLATE program
of the Wisconsin Package, version 9.1 (Genetics Computer Group,
Inc., Madison, Wis.) with a codon usage table compiled from the
chicken (Gallus gallus) ovalbumin, lysozyme, ovomucoid, and
ovotransferrin proteins. The DNA sequence was synthesized and
cloned into the 3' overhang T's of pCRII-TOPO (Invitrogen) by
Integrated DNA Technologies, Coralville, Iowa, on a contractual
basis. The EPO coding sequence was then removed from pEpoMM with
Hind III and Fse I, purified from a 0.8% agarose-TAE Gel, and
ligated to Hind III and Fse I digested, alkaline
phosphatase-treated pCMV-IFNMM. The resulting plasmid was
pAVIJCR-A137.43.2.2 which contained the EPO coding sequence
controlled by the cytomegalovirus immediate early promoter/enhancer
and SV40 polyA site. The plasmid pAVIJCR-A137.43.2.2 was digested
with Nco I and Fse I and the appropriate fragment ligated to an Nco
I and Fse I-digested fragment of pMDOTIFN to obtain
pAVIJCR-A137.87.2.1 which contained EPO driven by the MDOT
promoter. In order to clone the EPO coding sequence controlled by
the MDOT promoter into the NLB retroviral plasmid, the plasmids
pALVMDOTIFN and pAVIJCR-A137.87.2.1 were digested with Kpn I and
Fse I. Appropriate DNA fragments were purified on a 0.8%
agarose-TAE gel, then ligated and transformed into DH5.alpha.
cells. The resulting plasmid was pNLB-MDOT-EPO (a.k.a.
pAVIJCR-A145.27.2.2).
Example 11
Production of Transgenic Chickens and Fully Transgenic G1 Chickens
Expressing EPO
[0149] Production of NLB-MDOT-EPO transduction particles were
performed as described for NLB-CMV-BL (see Example 2).
Approximately 300 White Leghorn eggs were windowed according to the
Speksnijder procedure (U.S. Pat. No. 5,897,998), then injected with
.about.7.times.10.sup.4 transducing particles per egg. Eggs hatched
21 days after injection, and human EPO levels were measured by EPO
ELISA from serum samples collected from chicks one week after
hatch.
[0150] In order to screen for G.sub.0 roosters which contained the
EPO transgene in their sperm, DNA was extracted from rooster sperm
samples by Chelex-100 extraction (Walsh et al., 1991). DNA samples
were then subjected to Taqman.TM. analysis on a 7700 Sequence
Detector (Perkin Elmer) using the "neo for-1"
(5'-TGGATTGCACGCAGGTTCT-3') and "neo rev-1"
(5'-GTGCCCAGTCATAGCCGAAT-3') primers and FAM labeled NEO-PROBE1
(5'-CCTCTCCACCCAAGCGGCCG-3') to detect the transgene. Eight G.sub.0
roosters with the highest levels of the transgene in their sperm
samples were bred to nontransgenic SPAFAS (White Leghorn) hens by
artificial insemination. Blood DNA samples were screened for the
presence of the transgene by Taqman.TM. analysis as described
above.
[0151] Out of 1,054 offspring, 16 chicks were found to be
transgenic (G1 avians). Chick serum was tested for the presence of
human EPO by EPO ELISA, and EPO was present at .about.70
nanogram/ml (ng/ml). Egg white in eggs from G1 hens was also tested
for the presence of human EPO by EPO ELISA and found to contain
human EPO at .about.70 ng/ml. The EPO present in eggs (i.e.,
derived from the optimized coding sequence of human EPO) was found
to be biologically active when tested on a human EPO responsive
cell line (HCD57 murine erythroid cells) in a cell culture
assay.
Example 12
Vector Construction for pNLB-CMV-IFN
[0152] Following the teachings of Example 1, a pNLB-CMV-IFN vector
was created (FIG. 8A), substituting an IFN encoding sequence for
the BL encoding sequence of Example 1.
[0153] An optimized coding sequence was created, wherein the most
frequently used codons for each particular amino acid found in the
egg white proteins ovalbumin, lysozyme, ovomucoid, and
ovotransferrin were used in the design of the optimized human
IFN-.alpha. 2b coding sequence that was inserted into vectors of
the instant invention. More specifically, the DNA sequence for
optimized human IFN-.alpha. 2b is based on the hen oviduct
optimized codon usage and was created using the BACKTRANSLATE
program of the Wisconsin Package, Version 9.1 (Genetics Computer
Group Inc., Madison, Wis.) with a codon usage table compiled from
the chicken (Gallus gallus) ovalbumin, lysozme, ovomucoid, and
ovotransferrin proteins. For example, the percent usage for the
four codons of the amino acid alanine in the four egg white
proteins is 34% for GCU, 31% for GCC, 26% for GCA, and 8% for GCG.
Therefore, GCU was used as the codon for the majority of alanines
in the optimized human IFN-.alpha. 2b coding sequence. The vectors
containing the gene for optimized human IFN-.alpha. 2b were used to
create transgenic avians that express transgenic poultry derived
interferon-.alpha. 2b (TPD IFN-.alpha. 2b) in their tissues and
eggs.
[0154] The template and primer oligonucleotides listed in Table 4
below were amplified by PCR with Pfu polymerase (Stratagene, La
Jolla, Calif.) using 20 cycles of 94.degree. C. for 1 min.;
50.degree. C. for 30 sec.; and 72.degree. C. for 1 min. and 10 sec.
PCR products were purified from a 12% polyacrylamide-TBE gel by the
"crush and soak" method (Maniatis et al. 1982), then combined as
templates in an amplification reaction using only IFN-1 and IFN-8
as primers (see Table 4). The resulting PCR product was digested
with Hind III and Xba I and gel purified from a 2% agarose-TAE gel,
then ligated into Hind III and Xba I digested, alkaline
phosphatase-treated pBluescript KS (Stratagene), resulting in the
plasmid pBluKSP-IFNMagMax. Both strands were sequenced by cycle
sequencing on an ABI PRISM 377 DNA Sequencer (Perkin-Elmer, Foster
City, Calif.) using universal T7 or T3 primers. Mutations in
pBluKSP-IFN derived from the original oligonucleotide templates
were corrected by site-directed mutagenesis with the Transformer
Site-Directed Mutagenesis Kit (Clontech, Palo Alto, Calif.). The
IFN coding sequence was then removed from the corrected pBluKSP-IFN
with Hind III and Xba 1, purified from a 0.8% agarose-TAE Gel, and
ligated to Hind III and Xba I digested, alkaline
phosphatase-treated pCMV-BetaLa-3B-dH. The resulting plasmid was
pCMV-IFN which contained an IFN coding sequence controlled by the
cytomegalovirus immediate early promoter/enhancer and SV40 polyA
site. In order to clone the IFN coding sequence controlled by the
CMV promoter/enhancer into the NLB retroviral plasmid, pCMV-IFN was
first digested with ClaI and XbaI, then both ends were filled in
with Klenow fragment of DNA polymerase (New England BioLabs,
Beverly, Mass.). pNLB-adapter was digested with NdeI and KpnI, and
both ends were made blunt by T4 DNA polymerase (New England
BioLabs). Appropriate DNA fragments were purified on a 0.8%
agarose-TAE gel, then ligated and transformed into DH5.alpha.
cells. The resulting plasmid was pNLB-adapter-CMV-IFN. This plasmid
was then digested with MluI and partially digested with BlpI and
the appropriate fragment was gel purified. pNLB-CMV-EGFP was
digested with MluI and BlpI, then alkaline-phosphatase treated and
gel purified. The MluI/BlpI partial fragment of
pNLB-adapter-CMV-IFN was ligated to the large fragment derived from
the MluI/BlpI digest of pNLB-CMV-EGFP creating pNLB-CMV-IFN.
4TABLE 4 Oligonucleotides used for IFN Gene Synthesis Primer Primer
Template Sequence of Template 1 Sequence of Primer 1 2 Sequence of
Primer 2 IFN-A 5'ATGGCTTTGACCTTTGCCT IFN-1 5'CCCAAGCTTTCACCA IFN-2
5'CTGTGGGTCTGAGGC TACTGGTGGCTCTCCTGGTG TGGCTTTGACCTTTGCC AGAT3'
CTGAGCTGCAAGAGCAGCT TT3' GCTCTGTGGGCTGCGATCTG CCTCA3' IFN-B
5'GACCCACAGCCTGGGCAG IFN-2b 5'ATCTGCCTCAGACCC IFN-3b
5'AACTCCTCTTGAGGA CAGGAGGACCCTGATGCTG ACAG3' AAGCCAAAATC3'
CTGGCTCAGATGAGGAGAA TCAGCCTGTITAGCTGCCTG AAGGATAGGCACGATTTTG
GCTTT3' IFN-C 5'CTCAAGAGGAGTTTGGCA IFN-3c 5'GATTTTGGCTTTCCTC IFN-4
5'ATCTGCTGGATCATC ACCAGTTTCAGAAGGCTGA AAGAGGAGTT3' TCGTGC3'
GACCATCCCTGTGCTGCAC GAGATG3' IFN-D 5'ATCCAGCAGATCTTTAAC IFN-4b
5'GCACGAGATGATCCA IFN-5 5'ATCGTTCAGCTGCTG CTGTTTAGCACCAAGGATA
GCAGAT3' GTACA3' GCAGCGCTGCTTGGGATGA GACCCTGCTGGATAAGTTTT
ACACCGAGCTGTACCAGCA 3' IFN-E 5'GCTGAACGATCTGGAGGC IFN-5b
5'TGTACCAGCAGCTGA IFN-6 5'CCTCACAGCCAGGAT TTGCGTGATCCAGGGCGTG
ACGAT3' GCTAT3' GGCGTGACCGAGACCCCTC TGATGAAGGAGGATAGCAT CCT3' IFN-F
5'GGCTGTGAGGAAGTACTT IFN-6b 5'ATAGCATCCTGGCTG IFN-7
5'ATGATCTCAGCCCTC TCAGAGGATCACCCTGTAC TGAGG 3' ACGAC3'
CTGAAGGAGAAGAAGTACA GCCCTTGCGCTTGGGAAGT CGTGAGGG3' IFN-G
5'CTGAGATCATGAGGAGCT IFN-7b 5'GTCGTGAGGGCTGAG IFN-8
5'TGCTCTAGACTTTTTA TTAGCCTGAGCACCAACCT ATCAT 3' CTCCTTAGACCTCAAGC
GCAAGAGAGCTTGAGGTCT TCT3' AAGGAGTAA3'
Example 13
Production of Transgenic Chickens and Fully Transgenic G1 Chickens
Expressing IFN
[0155] Transduction particles of pNLB-CMV-IFN were produced
following the procedures of Example 2. Approximately 300 White
Leghorn (strain Line 0) eggs were windowed according to the
Speksnijder procedure (U.S. Pat. No. 5,897,998), then injected with
.about.7.times.10.sup.4 transducing particles per egg. Eggs hatched
21 days after injection, and human IFN levels were measured by IFN
ELISA from serum samples collected from chicks one week after
hatch.
[0156] In order to screen for G.sub.0 roosters which contained the
IFN transgene in their sperm, DNA was extracted from rooster sperm
samples by Chelex-100 extraction (Walsh et al., 1991). DNA samples
were then subjected to Taqman.TM. analysis on a 7700 Sequence
Detector (Perkin Elmer) using the "neo for-1"
(5'-TGGATTGCACGCAGGTTCT-3') and "neo rev-1"
(5'-GTGCCCAGTCATAGCCGAAT-3') primers and FAM labeled NEO-PROBE1
(5'-CCTCTCCACCCAAGCGGCCG-3') to detect the transgene. Three G.sub.0
roosters with the highest levels of the transgene in their sperm
samples were bred to nontransgenic SPAFAS (White Leghorn) hens by
artificial insemination
[0157] Blood DNA samples were screened for the presence of the
transgene by Taqman.TM. analysis as described above. Out of 1,597
offspring, one rooster was found to be transgenic (a.k.a.
"Alphie"). Alphie's serum was tested for the presence of hIFN by
hIFN ELISA, and hIFN was present at 200 ng/ml.
[0158] Alphie's sperm was used for artificial insemination of
nontransgenic SPAFAS (White Leghorn) hens. 106 out of 202
(.about.52%) offspring contained the transgene as detected by
Taqman.TM. analysis. These breeding results followed a Mendelian
inheritance pattern and indicated that Alphie is transgenic.
Example 14
Carbohydrate Analysis of Transgenic Poultry Derived
Interferon-.alpha. 2b (TPD IFN-.alpha. 2b)
[0159] Experimental evidence revealed a new glycosylation pattern
in interferon-.alpha. 2b derived from avians (i.e., TPD IFN-.alpha.
2b). TPD IFN-.alpha. 2b was found to contain two new glyco forms
(bands 4 and 5 are .alpha.-Gal extended disaccharides; see FIG. 9)
not normally seen in human peripheral blood leukocyte derived
interferon-.alpha. 2b (PBL IFN-.alpha. 2b) or natural human
interferon-.alpha. 2b (natural hIFN). TPD IFN-.alpha. 2b was also
found to contain O-linked carbohydrate structures that are similar
to human PBL IFN-.alpha. 2b and was more efficiently produced in
chickens then the human form.
[0160] The coding sequence for human IFN-.alpha. 2b was optimized
(Example 12, supra) resulting in a recombinant IFN-.alpha. 2b
coding sequence. TPD IFN-.alpha. 2b was then produced in chickens
(Example 13, supra). A carbohydrate analysis, including a
monosaccharide analysis and FACE analysis, revealed the sugar
make-up or novel glycosylation pattern of the protein. As such, TPD
IFN-.alpha. 2b showed the following monosaccharide residues:
N-Acetyl-Galactosamine (NAcGal), Galactose (Gal),
N-Acetyl-Glucosamine (NAcGlu), and Sialic acid (SA). No N-linked
glycosylation was found in TPD IFN-.alpha. 2b. Instead, TPD
IFN-.alpha. 2b was found to be O-glycosylated at Thr-106. This type
of glycosylation is similar to human IFN-.alpha. 2, wherein the Thr
residue at position 106 is unique to IFN-.alpha. 2. In addition,
TPD IFN-.alpha. 2b was found to have no mannose residues. A FACE
analysis revealed 6 bands (FIG. 9) that represent various sugar
residues, wherein bands 1, 2 and 3 are un-sialyated,
mono-sialyated, and di-sialyated, respectively (FIG. 10). The
sialic acid (SA) linkage is alpha 2-3 to Galactose (Gal) and alpha
2-6 to N-Acetyl-Galactosamine (NAcGal). Band 6 represents an
un-sialyated tetrasaccharide. Bands 4 and 5 were found to be
alpha-Galactose (alpha-Gal) extended disaccharides that are not
seen in human PBL IFN-.alpha. 2b. FIG. 10 shows the comparison of
TPD IFN-.alpha. 2b (egg white hIFN) and human PBL IFN-.alpha. 2b
(natural hIFN). Minor bands were present between bands 3 and 4 and
between bands 4 and 5 in TPD IFN-.alpha. 2b (vide infra).
[0161] The protein was found to be O-glycosylated at Thr-106 with
specific residues, such as: 3
[0162] wherein Gal=Galactose,
[0163] NAcGal=N-Acetyl-Galactosamine,
[0164] NAcGlu=N-Acetyl-Glucosamine, and
[0165] SA=Sialic Acid.
[0166] The percentages were as follows:
[0167] (i) Gal-NAcGal- is about 20%
[0168] (ii) SA-Gal-NAcGal- is about 29% 4
[0169] Minor bands were present between bands 3 and 4 and between
bands 4 and 5 which account for about 17% in TPD IFN-.alpha.
2b.
Example 15
Expression of MAbs from Plasmid Transfection and Retroviral
Transduction Using the EMCV IRES in Avian Cells
[0170] The light chain (LC) and heavy chain (HC) of a human
monoclonal antibody were expressed from a single vector,
pCMV-LC-emcvIRES-HC, by placement of an IRES from the
encephalomyocarditis virus (EMCV) (see also Jang et al. (1988) "A
segment of the 5' nontranslated region of encephalomyocarditis
virus RNA directs internal entry of ribosomes during in vitro
translation" J. Virol. 62:2636-2643) between the LC and HC coding
sequences. Transcription was driven by the CMV promoter.
[0171] In order to test expression of monoclonal antibodies from
two separate vectors, the LC or HC linked to the CMV promoter were
cotransfected into LMH/2a cells, an estrogen-responsive, chicken
hepatocyte cell line (see also Binder et al. (1990) "Expression of
endogenous and transfected apolipoprotein II and vitellogenin II
genes in an estrogen responsive chicken liver cell line" Mol.
Endocrinol. 4:201-208). Contransfection of pCMV-LC and pCMV-HC
resulted in 392 ng/ml of MAbs determined by a MAB ELISA whereas
transfection of pCMV-LC-emcvIRES-HC resulted in 185 ng/ml of
MAb.
[0172] The CMV-LC-emcv-HC cassette was inserted in a retroviral
vector based on the Moloney murine leukemia virus (MLV), creating
pL-CMV-LC-emcvIRES-HC-RN-BG. LMH cells (see also Kawaguchi et al.
(1987) "Establishment and characterization of a chicken
hepatocellular carcinoma cell line, LMH" Cancer Res. 47:4460-4464),
the parent line of LMH/2a, were used as target cells because they
are not neomycin resistant. LMH cells were transduced with the
L-CMV-LC-emcvIRES-HC-RN-BG retroviral vector and selected with
neomycin and passaged for several weeks. LMH cells were separately
transduced and neomycin selected with the parent MLV vector, LXRN.
Media from LXRN cells were negative for MAb, whereas media from the
L-CMV-LC-emcvIRES-HC-RN-BG-transduced cells contained 22 ng/ml of
MAb.
[0173] All documents (e.g., U.S. patents, U.S. patent applications,
publications) cited in the above specification are herein
incorporated by reference. Various modifications and variations of
the present invention will be apparent to those skilled in the art
without departing from the scope and spirit of the invention.
Although the invention has been described in connection with
specific preferred embodiments, it should be understood that the
invention as claimed should not be unduly limited to such specific
embodiments. Indeed, various modifications of the described modes
for carrying out the invention which are obvious to those skilled
in the art are intended to be within the scope of the following
claims.
Sequence CWU 1
1
31 1 498 DNA Artificial Sequence TPD IFN alpha 2b 1 tgcgatctgc
ctcagaccca cagcctgggc agcaggagga ccctgatgct gctggctcag 60
atgaggagaa tcagcctgtt tagctgcctg aaggataggc acgattttgg ctttcctcaa
120 gaggagtttg gcaaccagtt tcagaaggct gagaccatcc ctgtgctgca
cgagatgatc 180 cagcagatct ttaacctgtt tagcaccaag gatagcagcg
ctgcttggga tgagaccctg 240 ctggataagt tttacaccga gctgtaccag
cagctgaacg atctggaggc ttgcgtgatc 300 cagggcgtgg gcgtgaccga
gacccctctg atgaaggagg atagcatcct ggctgtgagg 360 aagtactttc
agaggatcac cctgtacctg aaggagaaga agtacagccc ctgcgcttgg 420
gaagtcgtga gggctgagat catgaggagc tttagcctga gcaccaacct gcaagagagc
480 ttgaggtcta aggagtaa 498 2 165 PRT Artificial Sequence TPD IFN
alpha 2b 2 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr
Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser
Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu
Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu
His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys
Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe
Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys
Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110
Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115
120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu
Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 3 579 DNA
Artificial Sequence TPD EPO 3 atgggcgtgc acgagtgccc tgcttggctg
tggctgctct tgagcctgct cagcctgcct 60 ctgggcctgc ctgtgctggg
cgctcctcca aggctgatct gcgatagcag ggtgctggag 120 aggtacctgc
tggaggctaa ggaggctgag aacatcacca ccggctgcgc tgagcactgc 180
agcctgaacg agaacatcac cgtgcctgat accaaggtga acttttacgc ttggaagagg
240 atggaggtgg gccagcaggc tgtggaggtg tggcagggcc tggctctgct
gagcgaggct 300 gtgctgaggg gccaggctct gctggtgaac agctctcagc
cttgggagcc tctgcagctg 360 cacgtggata aggctgtgag cggcctgaga
agcctgacca ccctgctgag ggctctgggc 420 gctcagaagg aggctatcag
ccctccagat gctgcaagcg ctgcccctct gaggaccatc 480 accgctgata
cctttaggaa gctgtttagg gtgtacagca actttctgag gggcaagctg 540
aagctgtaca ccggcgaggc ttgcaggacc ggcgatagg 579 4 193 PRT Artificial
Sequence TPD EPO 4 Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu
Leu Leu Ser Leu 1 5 10 15 Leu Ser Leu Pro Leu Gly Leu Pro Val Leu
Gly Ala Pro Pro Arg Leu 20 25 30 Ile Cys Asp Ser Arg Val Leu Glu
Arg Tyr Leu Leu Glu Ala Lys Glu 35 40 45 Ala Glu Asn Ile Thr Thr
Gly Cys Ala Glu His Cys Ser Leu Asn Glu 50 55 60 Asn Ile Thr Val
Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg 65 70 75 80 Met Glu
Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu 85 90 95
Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser 100
105 110 Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser
Gly 115 120 125 Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala
Gln Lys Glu 130 135 140 Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala
Pro Leu Arg Thr Ile 145 150 155 160 Thr Ala Asp Thr Phe Arg Lys Leu
Phe Arg Val Tyr Ser Asn Phe Leu 165 170 175 Arg Gly Lys Leu Lys Leu
Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp 180 185 190 Arg 5 19 DNA
Artificial Sequence neo for-1 primer 5 tggattgcac gcaggttct 19 6 20
DNA Artificial Sequence neo rev-1 primer 6 gtgcccagtc atagccgaat 20
7 20 DNA Artificial Sequence NEO-PROBE1 7 cctctccacc caagcggccg 20
8 83 DNA Artificial Sequence IFN-A primer 8 atggctttga cctttgcctt
actggtggct ctcctggtgc tgagctgcaa gagcagctgc 60 tctgtgggct
gcgatctgcc tca 83 9 34 DNA Artificial Sequence IFN-1 primer 9
cccaagcttt caccatggct ttgacctttg cctt 34 10 19 DNA Artificial
Sequence IFN-2 primer 10 ctgtgggtct gaggcagat 19 11 100 DNA
Artificial Sequence IFN-B primer 11 gacccacagc ctgggcagca
ggaggaccct gatgctgctg gctcagatga ggagaatcag 60 cctgtttagc
tgcctgaagg ataggcacga ttttggcttt 100 12 19 DNA Artificial Sequence
IFN-2b primer 12 atctgcctca gacccacag 19 13 26 DNA Artificial
Sequence IFN-3b primer 13 aactcctctt gaggaaagcc aaaatc 26 14 62 DNA
Artificial Sequence IFN-C primer 14 ctcaagagga gtttggcaac
cagtttcaga aggctgagac catccctgtg ctgcacgaga 60 tg 62 15 26 DNA
Artificial Sequence IFN-3c primer 15 gattttggct ttcctcaaga ggagtt
26 16 21 DNA Artificial Sequence IFN-4 primer 16 atctgctgga
tcatctcgtg c 21 17 95 DNA Artificial Sequence IFN-D primer 17
atccagcaga tctttaacct gtttagcacc aaggatagca gcgctgcttg ggatgagacc
60 ctgctggata agttttacac cgagctgtac cagca 95 18 21 DNA Artificial
Sequence IFN-4b primer 18 gcacgagatg atccagcaga t 21 19 20 DNA
Artificial Sequence IFN-5 primer 19 atcgttcagc tgctggtaca 20 20 78
DNA Artificial Sequence IFN-E primer 20 gctgaacgat ctggaggctt
gcgtgatcca gggcgtgggc gtgaccgaga cccctctgat 60 gaaggaggat agcatcct
78 21 20 DNA Artificial Sequence IFN-5b primer 21 tgtaccagca
gctgaacgat 20 22 20 DNA Artificial Sequence IFN-6 primer 22
cctcacagcc aggatgctat 20 23 83 DNA Artificial Sequence IFN-F primer
23 ggctgtgagg aagtactttc agaggatcac cctgtacctg aaggagaaga
agtacagccc 60 ttgcgcttgg gaagtcgtga ggg 83 24 20 DNA Artificial
Sequence IFN-6b primer 24 atagcatcct ggctgtgagg 20 25 20 DNA
Artificial Sequence IFN-7 primer 25 atgatctcag ccctcacgac 20 26 65
DNA Artificial Sequence IFN-G primer 26 ctgagatcat gaggagcttt
agcctgagca ccaacctgca agagagcttg aggtctaagg 60 agtaa 65 27 20 DNA
Artificial Sequence IFN-7b primer 27 gtcgtgaggg ctgagatcat 20 28 36
DNA Artificial Sequence IFN-8 primer 28 tgctctagac tttttactcc
ttagacctca agctct 36 29 69 DNA Artificial Sequence Lysozyme signal
sequence 29 ccaccatggg gtctttgcta atcttggtgc tttgcttcct gccgctagct
gccttagggc 60 cctctagag 69 30 671 DNA Artificial Sequence MDOT
promoter linked to IFN-MM CDS 30 atcgataggt accgggcccc ccctcgaggt
gaatatccaa gaatgcagaa ctgcatggaa 60 agcagagctg caggcacgat
ggtgctgagc cttagctgct tcctgctggg agatgtggat 120 gcagagacga
atgaaggacc tgtcccttac tcccctcagc attctgtgct atttagggtt 180
ctaccagagt ccttaagagg tttttttttt ttttggtcca aaagtctgtt tgtttggttt
240 tgaccactga gagcatgtga cacttgtctc aagctattaa ccaagtgtcc
agccaaaatc 300 gatgtcacaa cttgggaatt ttccatttga agccccttgc
aaaaacaaag agcaccttgc 360 ctgctccagc tcctggctgt gaagggtttt
ggtgccaaag agtgaaaggc ttcctaaaaa 420 tgggctgagc cggggaaggg
gggcaacttg ggggctattg agaaacaagg aaggacaaac 480 agcgttaggt
cattgcttct gcaaacacag ccagggctgc tcctctataa aaggggaaga 540
aagaggctcc gcagccatca cagacccaga ggggacggtc tgtgaatcaa gctttcacca
600 tggctttgac ctttgcctta ctggtggctc tcctggtgct gagctgcaag
agcagctgct 660 cgtgggttgc g 671 31 24 PRT Artificial Sequence MDOT
promoter linked to IFN-MM CDS 31 Met Ala Leu Thr Phe Ala Leu Leu
Val Ala Leu Leu Val Leu Ser Cys 1 5 10 15 Lys Ser Ser Cys Ser Trp
Val Ala 20
* * * * *