U.S. patent application number 11/774954 was filed with the patent office on 2008-08-28 for modified pituitary gland development in offspring from expectant mother animals treated with growth hormone releasing hormone therapy.
This patent application is currently assigned to Baylor College of Medicine. Invention is credited to Ruxandra Draghia-Akli, Amir Khan.
Application Number | 20080207543 11/774954 |
Document ID | / |
Family ID | 27734536 |
Filed Date | 2008-08-28 |
United States Patent
Application |
20080207543 |
Kind Code |
A1 |
Draghia-Akli; Ruxandra ; et
al. |
August 28, 2008 |
MODIFIED PITUITARY GLAND DEVELOPMENT IN OFFSPRING FROM EXPECTANT
MOTHER ANIMALS TREATED WITH GROWTH HORMONE RELEASING HORMONE
THERAPY
Abstract
The intramuscular electroporated injection of a
protease-resistant growth hormone-releasing hormone ("GHRH") cDNA
into rat dams at 16 days of gestation resulted in the enhanced
long-term growth of the FI offspring. The offspring were
significantly heavier by one week of age and the difference was
sustained to 10 weeks of age. Consistent with their augmented
growth, plasma IGF-I concentration of the FI progeny was increased
significantly. The pituitary gland of the offspring was
significantly heavier, and contained an increased number of
somatotropes (cells producing GH) and lactotrophs
(prolactin-secreting cells), and is indicative of an alteration in
cell lineages. These unique findings demonstrate that enhanced GHRH
expression in pregnant dams can result in intergenerational growth
promotion, by altering development of the pituitary gland in the
offspring.
Inventors: |
Draghia-Akli; Ruxandra;
(Houston, TX) ; Khan; Amir; (The Woodlands,
TX) |
Correspondence
Address: |
Pepper Hamilton LLP
400 Berwyn Park, 899 Cassatt Road
Berwyn
PA
19312-1183
US
|
Assignee: |
Baylor College of Medicine
Houston
TX
|
Family ID: |
27734536 |
Appl. No.: |
11/774954 |
Filed: |
July 9, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10359919 |
Feb 6, 2003 |
7250405 |
|
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11774954 |
|
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60355566 |
Feb 7, 2002 |
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Current U.S.
Class: |
514/44R ;
435/455; 435/461 |
Current CPC
Class: |
A61P 5/02 20180101; A61K
38/00 20130101; A61K 48/005 20130101; A61P 5/06 20180101; C07K
14/60 20130101; A61K 48/00 20130101 |
Class at
Publication: |
514/44 ; 435/455;
435/461 |
International
Class: |
A61K 31/7052 20060101
A61K031/7052; C12N 15/87 20060101 C12N015/87; A61P 5/06 20060101
A61P005/06 |
Claims
1. A method of changing the pituitary lineage in an offspring from
a female subject comprising: delivering a nucleic acid expression
construct into cells of the female subject; wherein, the delivering
is completed prior to or during a gestation period of the
offspring; the nucleic acid expression construct comprises a
promoter; a nucleotide sequence a growth-hormone-releasing-hormone
("GHRH") or functional biological equivalent thereof; and a 3'
untranslated region; and delivering is completed under conditions
wherein expression of the nucleotide sequence results in the
changing the pituitary lineage in the offspring;
2. The method of claim 1, wherein the delivering of the nucleic
acid expression construct into the cells of the female subject
comprises electroporation.
3. The method of claim 1, wherein the delivering of the nucleic
acid expression construct into the cells of the female subject
comprises use of a viral vector, a carrier, a parenteral route, or
a combination thereof.
4. The method of claim 1, wherein the cells of the female subject
comprise somatic cells, stem cells, or germ cells.
5. The method of claim 1, wherein the promoter comprises a
synthetic myogenic promoter.
6. The method of claim 1, wherein the 3' untranslated region
comprises a human growth hormone ("hGH") 3' untranslated region
(SEQ ID#8).
7. The method of claim 1, wherein the nucleic acid expression
construct comprises pSP-HV-GHRH (SEQ ID#11), or its functional
nucleic acid equivalent.
8. The method of claim 1, wherein the nucleic acid expression
construct is a TI-GHRH plasmid (SEQ ID#12), TV-GHRH Plasmid (SEQ
ID#13), 15/27/28 GHRH plasmid (SEQ ID#14), pSP-wt-GHRH plasmid (SEQ
ID#15), or its functional nucleic acid equivalent.
9. (canceled)
10. The method of claim 1, wherein the delivering into the cells of
the female subject the nucleic acid expression construct initiates
expression of the encoded GHRH or a functional biological
equivalent thereof.
11. The method of claim 1, wherein the encoded GHRH or functional
biological equivalent thereof comprise expression in tissue
specific cells of the female subject.
12. The method of claim 1, wherein the tissue specific cells of the
female subject comprise muscle cells.
13. The method of claim 1, wherein the nucleic acid expression
construct further comprises a transfection-facilitating
polypeptide.
14. The method of claim 13, wherein the transfection-facilitating
polypeptide comprises a charged polypeptide.
15. The method of claim 13, wherein the transfection-facilitating
polypeptide comprises poly-L-glutamate.
16. The method of claim 1, wherein the nucleic acid sequence
encodes a growth-hormone-releasing-hormone ("GHRH") or a functional
biological equivalent thereof, wherein the GHRH is a biologically
active polypeptide; and wherein the functional biological
equivalent of GHRH is a polypeptide that has been engineered to
contain a distinct amino acid sequence while simultaneously having
similar or improved biologically activity when compared to the GHRH
polypeptide.
17. The method of claim 1, wherein the nucleic acid sequence
encodes a growth-hormone-releasing-hormone ("GHRH") or a functional
biological equivalent thereof, and wherein the GHRH or the
functional biological equivalent comprises an amino acid formula
(SEQ ID No: 6):
-X-.sub.1-X.sub.2-DAIFTNSYRKVL-X.sub.3-QLSARKLLQDI-X.sub.4-X.sub.5-RQQGER-
NQEQGA-OH wherein the formula has the following characteristics:
X.sub.1 is a D- or L-isomer of the amino acid tyrosine ("Y"), or
histidine ("H"); X.sub.2 is a D- or L-isomer of the amino acid
alanine ("A"), valine ("V"), or isoleucine ("I"); X.sub.3 is a D-
or L-isomer of the amino acid alanine ("A") or glycine ("G");
X.sub.4 is a D- or L-isomer of the amino acid methionine ("M"), or
leucine ("L"); X.sub.5 is a D- or L-isomer of the amino acid serine
("S") or asparagine ("N").
18. The method of claim 1, wherein the nucleic acid sequence
encodes a growth-hormone-releasing-hormone ("GHRH") or a functional
biological equivalent thereof, that facilitates growth hormone
("GH") secretion in the female subject.
19. The method of claim 1, wherein the nucleic acid expression
construct is introduced into the female subject in a single
administration.
20. The method of claim 1, wherein delivering of the nucleic acid
expression construct into cells of the female subject occurs during
the third trimester of gestation of the offspring.
21. The method of claim 1, further comprising the step of
administering to the female subject a ligand for a growth hormone
secretagogue receptor.
22. The method of claim 21, wherein the ligand is administered
orally.
23. (canceled)
24. The method of claim 5, wherein the delivering of the nucleic
acid expression construct into the cells of the female subject
comprises electroporation.
25. The method of claim 5, wherein the delivering of the nucleic
acid expression construct into the cells of the female subject
comprises use of a viral vector, a carrier, a parenteral route, or
a combination thereof.
26. The method of claim 5, wherein the cells of the female subject
comprise somatic cells, stem cells, or germ cells.
27. The method of claim 5, wherein the 3' untranslated region
comprises a human growth hormone ("hGH") 3' untranslated region
(SEQ ID#8).
28. The method of claim 5, wherein the nucleic acid expression
construct comprises pSP-HV-GHRH (SEQ ID#11), or its functional
nucleic acid equivalent.
29. The method of claim 5, wherein the nucleic acid expression
construct is a TI-GHRH plasmid (SEQ ID#12), TV-GHRH Plasmid (SEQ
ID#13), 15/27/28 GHRH plasmid (SEQ ID#14), pSP-wt-GHRH plasmid (SEQ
ID#15), or its functional nucleic acid equivalent.
30. The method of claim 5, wherein the delivering into the cells of
the female subject the nucleic acid expression construct initiates
expression of the encoded GHRH or a functional biological
equivalent thereof.
31-32. (canceled)
33. The method of claim 5, wherein the nucleic acid expression
construct further comprises, an addition of a
transfection-facilitating polypeptide with nucleic acid expression
construct.
34. The method of claim 33, wherein the transfection-facilitating
polypeptide comprises a charged polypeptide.
35. The method of claim 33, wherein the transfection-facilitating
polypeptide comprises poly-L-glutamate.
36. The method of claim 5, wherein the nucleic acid sequence
encodes a growth-hormone-releasing-hormone ("GHRH") or a functional
biological equivalent thereof, wherein the GHRH is a biologically
active polypeptide; and wherein the functional biological
equivalent of GHRH is a polypeptide that has been engineered to
contain a distinct amino acid sequence while simultaneously having
similar or improved biologically activity when compared to the GHRH
polypeptide.
37. The method of claim 5, wherein the nucleic acid sequence
encodes a growth-hormone-releasing-hormone ("GHRH") or a functional
biological equivalent thereof, and wherein the GHRH or the
functional biological equivalent comprises an amino acid formula
(SEQ ID No: 6):
-X-.sub.1-X.sub.2-DAIFTNSYRKVL-X.sub.3-QLSARKLLQDI-X.sub.4-X.sub.5-RQQGER-
NQEQGA-OH wherein the formula has the following characteristics:
X.sub.1 is a D- or L-isomer of the amino acid tyrosine ("Y"), or
histidine ("H"); X.sub.2 is a D- or L-isomer of the amino acid
alanine ("A"), valine ("V"), or isoleucine ("I"); X.sub.3 is a D-
or L-isomer of the amino acid alanine ("A") or glycine ("G");
X.sub.4 is a D- or L-isomer of the amino acid methionine ("M"), or
leucine ("L"); X.sub.5 is a D- or L-isomer of the amino acid serine
("S") or asparagine ("N").
38. The method of claim 5, wherein the nucleic acid sequence
encodes a growth-hormone-releasing-hormone ("GHRH") or a functional
biological equivalent thereof, that facilitates growth hormone
("GH") secretion in the female subject.
39. The method of claim 5, wherein the nucleic acid expression
construct is introduced into the female subject in a single
administration.
40. The method of claim 5, wherein delivering of the nucleic acid
expression construct into cells of the female subject occurs during
the third trimester of gestation of the offspring.
41. The method of claim 5, further comprising the step of
administering to the female subject a ligand for a growth hormone
secretagogue receptor.
42. The method of claim 5, wherein the ligand is administered
orally.
43-84. (canceled)
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/355,566, entitled "MODIFIED PITUITARY GLAND
DEVELOPMENT IN OFFSPRING FROM EXPECTANT MOTHER ANIMALS TREATED WITH
GROWTH HORMONE RELEASING HORMONE THERAPY," filed on Feb. 7, 2002,
the entire content of which is hereby incorporated by
reference.
BACKGROUND
[0002] The present invention pertains to a plasmid-meditated gene
supplementation to alter pituitary development, and to increase
prolactin levels, in an offspring of a female subject. More
specifically, the present invention pertains to administering to a
female subject a nucleic acid expression construct that encodes
growth hormone releasing hormone ("GHRH") to alter the pituitary
development and pituitary hormone secretion (e.g. prolactin) in the
offspring from the female subject.
[0003] The pituitary gland is an important link between the nervous
system and the endocrine system. The pituitary gland is known to
release many hormones that affect growth, sexual development,
metabolism (e.g. protein, lipid and carbohydrate), glucocorticoids
and the reproductive system. The pituitary gland has also been
shown to release hormones that affect bone growth and regulate
activity in other hormone secreting glands. This invention relates
a method for altering pituitary gland development in offspring from
female subjects that have been treated with a nucleic acid
construct that encodes a growth hormone releasing hormone ("GHRH")
or functional biological equivalent. The expression of the GHRH or
biological equivalent thereof is regulated by a tissue specific
promoter (e.g. a myogenic promoter). When female subjects are
treated with the nucleic acid construct that encodes GHRH, many
physiological changes occur in the female subject directly.
However, when female subjects are treated with the GHRH construct
prior to, or during a gestation period, the offspring from these
treated female subjects undergo similar physiological changes. For
example, the subsequent expression and ensuing release of GHRH or
biological equivalent thereof by the modified cells in the female
subject results in the altered development of the pituitary gland
in their offspring. Additionally, hormones secreted by the
pituitary gland are increased in offspring from treated female
subjects when compared to the offspring from control treated female
subjects. More specifically, the pituitary gland is increased in
sized and the levels of the multi functional hormone prolactin is
elevated utilizing this method.
[0004] The pituitary gland has two distinct parts, the anterior and
the posterior lobes, each of which releases different hormones. The
pituitary gland appears to be subservient in part to the
hypothalamus. Pituitary gland development, including regulation and
differentiation of somatotrophs, depends upon paracrine processes
within the pituitary itself and involves several growth factors and
neuropeptides. Secretion of growth hormone ("GH") is stimulated by
the natural GH secretagogue, called growth hormone releasing
hormone ("GHRH"), and inhibited by somatostatin ("SS"). The central
role of growth hormone ("GH") is controlling somatic growth in
humans and other vertebrates, and the physiologically relevant
pathways that regulate GH secretion from the pituitary are well
known. For example, the GH production pathway is composed of a
series of interdependent genes whose products are required for
normal growth. The GH pathway genes include: (1) ligands, such as
GH and insulin-like growth factor-I ("IGF-I"); (2) transcription
factors such as prophet of pit 1, or prop 1, and pit 1: (3)
stimulatory and inhibitory factors, such as growth hormone
releasing hormone ("GHRH") and somatostatin ("SS"), respectively;
and (4) receptors, such as GHRH receptor ("GHRH-R") and the GH
receptor ("GH-R"). These genes are expressed in different organs
and tissues, including but not limited to the hypothalamus,
pituitary, liver, and bone. Effective and regulated expression of
the GH pathway is essential for optimal linear growth, as well as
homeostasis of carbohydrate, protein, and fat metabolism. GH
synthesis and secretion from the anterior pituitary is stimulated
by GHRH and inhibited by somatostatin, which are both hypothalamic
hormones. GH stimulates production of IGF-I, primarily in the
liver, and other target organs. IGF-I and GH, in tuni, feedback on
the hypothalamus and pituitary to inhibit GHRH and GH release. GH
elicits both direct and indirect actions on peripheral tissues, the
indirect effects being mediated mainly by IGF-I.
[0005] The immune function is modulated by IGF-I, which has two
major effects on B cell development: potentiation and maturation,
and as a B-cell proliferation cofactor that works together with
interleukin-7 ("IL-7"). These activities were identified through
the use of anti-IGF-I antibodies, antisense sequences to IGF-I, and
the use of recombinant IGF-I to substitute for the activity. There
is evidence that macrophages are a rich source of IGF-I. The
treatment of mice with recombinant IGF-I confirmed these
observations as it increased the number of pre-B and mature B cells
in bone marrow. The mature B cell remained sensitive to IGF-I as
immunoglobulin production was also stimulated by IGF-I in vitro and
in vivo.
[0006] The production of recombinant proteins in the last 2 decades
provided a useful tool for the treatment of many diverse
conditions. For example, recombinant GH administration has been
used to treat GH-deficiencies in short stature children, or as an
anabolic agent in burn, sepsis, and as well as in the elderly and
AIDS patients. However, resistance to GH action has been reported
in malnutrition and infection. Long-term studies on transgenic
animals and in patients undergoing GH therapies have shown no
causal correlation between GH or IGF-I therapy and cancer
development. GH replacement therapy is widely used clinically, with
beneficial effects, but therapy is associated several
disadvantages: GH must be administered subcutaneously or
intramuscularly once a day to three times a week for months, or
usually years; insulin resistance and impaired glucose tolerance
can occur; accelerated bone epiphysis growth and closure has been
observed in pediatric patients (Blethen, S. L., et al. 1996).
[0007] In contrast, essentially no side effects have been reported
for recombinant GHRH therapies. Extracranially secreted GHRH, as
mature peptide or truncated molecules (as seen with pancreatic
islet cell tumors and variously located carcinoids) are often
biologically active and can even produce acromegaly (Esch, et al.,
1982; Thorner, et al., 1984). Administration of recombinant GHRH to
GH-deficient children or adult humans augments IGF-I levels,
increases GH secretion proportionally to the GHRH dose, yet still
invokes a response to bolus doses of recombinant GHRH (Bercu and
Walker, 1997). Thus, GHRH administration represents a more
physiological alternative of increasing subnormal GH and IGF-I
levels (Corpas, et al., 1993).
[0008] GH is released in a distinctive pulsatile pattern that has
profound importance for its biological activity (Argente, et al.,
1996). Secretion of GH is stimulated by the GHRH, and inhibited by
somatostatin, and both are hypothalamic hormones (Thorner, et al.,
1995). GH pulses are a result of GHRH secretion that is associated
with a diminution or withdrawal of somatostatin secretion. In
addition, the pulse generator mechanism is timed by GH-negative
feedback. The endogenous rhythm of GH secretion becomes entrained
to the imposed rhythm of exogenous GH administration. Effective and
regulated expression of the GH and insulin-like growth factor-I
("IGF-I") pathway is essential for optimal linear growth,
homeostasis of carbohydrate, protein, and fat metabolism, and for
providing a positive nitrogen balance (Murray, et al., 2000).
Numerous studies in humans, sheep or pigs showed that continuous
infusion with recombinant GHRH protein restores the normal GH
pattern without desensitizing GHRH receptors or depleting GH
supplies as this system is capable of feed-back regulation, which
is abolished in the GH therapies (Dubreuil, et al., 1990). Although
recombinant GHRH protein therapy entrains and stimulates normal
cyclical GH secretion with virtually no side effects, the short
half-life of GHRH in vivo requires frequent (one to three times a
day) intravenous, subcutaneous or intranasal (requiring 300-fold
higher dose) administration. Thus, as a chronic treatment,
recombinant GHRH administration is not practical.
[0009] Wild type GHRH has a relatively short half-life in the
circulatory system, both in humans (Frohman, et al., 1984) and in
farm animals. After 60 minutes of incubation in plasma, 95% of the
GHRH(1-44)NH2 is degraded, while incubation of the shorter (1-40)OH
form of the hormone, under similar conditions, shows only a 77%
degradation of the peptide after 60 minutes of incubation (Frohman,
et al., 1989). Incorporation of cDNA coding for a particular
protease-resistant GHRH analog in a gene transfer vector results in
a molecule with a longer half-life in serum, increased potency, and
provides greater GH release in plasmid-injected animals
(Draghia-Akli, et al., 1999, herein incorporated by reference).
Mutagenesis via amino acid replacement of protease sensitive amino
acids prolongs the serum half-life of the GHRH molecule.
Furthermore, the enhancement of biological activity of GHRH is
achieved by using super-active analogs that may increase its
binding affinity to specific receptors (Draghia-Akli, et al.,
1999).
[0010] Extracranially secreted GHRH, as processed protein species
GHRH(1-40) hydroxy or GHRH(1-44) amide or even as shorter truncated
molecules, are biological active (Thorner, et al., 1984). It has
been reported that a low level of GHRH (100 pg/ml) in the blood
supply stimulates GH secretion (Corpas, et al., 1993). Direct
plasmid DNA gene transfer is currently the basis of many emerging
gene therapy strategies and thus does not require viral genes or
lipid particles (Muramatsu, et al., 1998; Aihara and Miyazaki,
1998). Skeletal muscle is target tissue, because muscle fiber has a
long life span and can be transduced by circular DNA plasmids that
express over months or years in an immunocompetent host (Davis, et
al., 1993; Tripathy, et al., 1996). Previous reports demonstrated
that human GHRH cDNA could be delivered to muscle by an injectable
myogenic expression vector in mice where it transiently stimulated
GH secretion to a modes extent over a period of two weeks
(Draghia-Akli, et al., 1997).
[0011] Administering novel GHRH analog proteins (U.S. Pat. Nos.
5,847,066; 5,846,936; 5,792,747; 5,776,901; 5,696,089; 5,486,505;
5,137,872; 5,084,442, 5,036,045; 5,023,322; 4,839,344; 4,410,512,
RE33,699) or synthetic or naturally occurring peptide fragments of
GHRH (U.S. Pat. Nos. 4,833,166; 4,228,158; 4,228,156; 4,226,857;
4,224,316; 4,223,021; 4,223,020; 4,223,019) for the purpose of
increasing release of growth hormone have been reported. A GHRH
analog containing the following mutations have been reported (U.S.
Pat. No. 5,846,936): Tyr at position 1 to H is; Ala at position 2
to Val, Leu, or others; Asn at position 8 to Gln, Scr, or Thr; Gly
at position 15 to Ala or Leu; Met at position 27 to Nle or Leu; and
Ser at position 28 to Asn. The GHRH analog is the subject of U.S.
patent application Ser. No. 09/624,268 ("the '268 application"),
which teaches application of a GHRH analog containing mutations
that improve the ability to elicit the release of growth hormone.
In addition, the '268 application relates to the treatment of
growth deficiencies; the improvement of growth performance; the
stimulation of production of growth hormone in an animal at a
greater level than that associated with normal growth; and the
enhancement of growth utilizing the administration of growth
hormone releasing hormone analog and is herein incorporated by
reference.
[0012] U.S. Pat. No. 5,061,690 is directed toward increasing both
birth weight and milk production by supplying to pregnant female
mammals an effective amount of human GHRH or one of it analogs for
10-20 days. Application of the analogs lasts only throughout the
lactation period. However, multiple administrations are needed. A
co-pending disclosure regarding administration of the growth
hormone releasing hormone (or factor) as a DNA molecule, such as
with plasmid mediated therapy techniques has been disclosed (U.S.
patent application Ser. No. 10/021,403).
[0013] U.S. Pat. Nos. 5,134,120 ("the '120 patent") and 5,292,721
("the '721 patent") teach that by deliberately increasing growth
hormone in swine during the last 2 weeks of pregnancy through a 3
week lactation resulted in the newborn piglets having marked
enhancement of the ability to maintain plasma concentrations of
glucose and free fatty acids when fasted after birth. In addition,
the '120 and '721 patents teach that treatment of the sow during
lactation results in increased milk fat in the colostrum and an
increased milk yield. These effects are important in enhancing
survivability of newborn pigs and weight gain prior to weaning.
However, the '120 and '721 patents provide no teachings regarding
administration of the growth hormone releasing hormone ("GHRH") as
a DNA form.
[0014] Prolactin is a single-chain protein hormone closely related
to growth hormone. It is chiefly secreted by lactotrophs in the
anterior pituitary. However, prolactin is also synthesized and
secreted by a broad range of other cells in the body, most
prominently various immune cells, the brain and the decidua of the
pregnant uterus. Prolactin is also found in the serum of normal
females and males. Prolactin secretion is pulsatile and also shows
diurnal variation, with the serum concentration increasing during
sleep and the lowest level occurs about 3 hours after waking. The
secretion of prolactin is increased by stress and appears to be
dependent upon a women's estrogen status.
[0015] The conventional view of prolactin is that the mammary gland
is its major target organ, and stimulating mammary gland
development along with milk production define its major functions.
Although these views are true, such descriptions fail to convey an
accurate depiction of this multifunctional hormone. For example, it
is difficult to find a mammalian tissue that does not express
prolactin receptors, and although the anterior pituitary is the
major source of prolactin, the hormone is synthesized and secreted
in many other tissues. Overall, several hundred different actions
have been reported for prolactin in various species. Some of
prolactin's major effects are summarized below.
[0016] Prolactin's major known functions are attributed with
mammary gland development, milk production and reproduction. In the
1920's it was found that extracts of the pituitary gland, when
injected into virgin rabbits, induced milk production. Subsequent
research demonstrated that prolactin has two major roles in milk
production: [0017] Prolactin induces lobulo-alveolar growth of the
mammary gland, wherein the alveoli are the clusters of cells in the
mammary gland that actually secrete milk. [0018] Prolactin
stimulates lactogenesis or milk production after giving birth.
Prolactin, along with cortisol and insulin, act together to
stimulate transcription of the genes that encode milk proteins. The
critical role of prolactin in lactation has been established by
utilizing transgenic mice with targeted deletions in the prolactin
gene. Female mice that are heterozygous for the deleted prolactin
gene only produce about half the normal amount of prolactin, and
fail to lactate after their first pregnancy.
[0019] Prolactin is also important in several non-lactational
aspects of reproduction. For example, in some species (e.g.
rodents, dogs, skunks), prolactin is necessary for maintenance of
ovarian structures (i.e. corpora lutea) that secrete progesterone.
Mice that are homozygous for an inactivated prolactin gene and thus
incapable of secreting prolactin are infertile due to defects in
ovulation, fertilization, preimplantation development and
implantation. Prolactin also appears to have stimulatory effects in
some species on reproductive or maternal behaviors such as nest
building and retrieval of scattered young.
[0020] Prolactin also appears to elicit effects in the immune
system. For example, the prolactin receptor is widely expressed by
immune cells, and some types of lymphocytes synthesize and secrete
prolactin. These observations suggest that prolactin may act as an
autocrine or paracrine modulator of immune activity. Conversely,
mice with homozygous deletions of the prolactin gene fail to show
significant abnormalities in immune responses. A considerable
amount of research is in progress to delineate the role of
prolactin in normal and pathologic immune responses. However, the
significance of these potential functions remains poorly
understood.
[0021] Administering prolactin stimulating hormones, or prolactin
agonists (U.S. Pat. Nos. 5,605,885; and 5,872,127) for the purpose
of stimulating the immune system have been reported. The U.S. Pat.
No. 5,872,127 ("the '127 patent") filed by Cincotta in 1999
discloses methods for treating a disorder of the immune system or
an immunodeficiency state that comprise the steps of administering
to a patient an effective amount a serotonin agonist and at a
dopamine agonist, where the combination of the serotonin agonist
and the dopamine agonist are present in an amount effective to
treat a patient's immuno-compromised condition. The administration
of each of the agents is confined to a specific time of day that is
capable of adjusting the prolactin profile of the patient to
conform or to approach the standard human prolactin profile.
[0022] Additionally, the supplementation of the prolactin agonists
in U.S. Pat. No. 5,605,885 ("the '885 patent") disclose a method
for the stimulation of a suppressed or deficient immune system by
regulating the blood levels or activity of the hormone prolactin
directly. The '885 patent method comprises treating an
immunosuppressed subject with proteins, peptides and compounds that
have prolactin-like activity including, but not limited to,
prolactin, peptide sequences from prolactin that have
prolactin-like activity, growth hormone (a structurally similar and
biologically related hormone), or peptide sequences from growth
hormone which have prolactin-like activity, placental lactogens,
and any genetically engineered protein sequence which has
prolactin-like activity. However, neither the '885 and '127 patents
provide teachings regarding increasing prolactin levels by the
administration of the growth hormone releasing hormone ("GHRH") as
a DNA form.
[0023] In summary, the production of recombinant proteins in the
last 2 decades provides a useful tool for the treatment of many
diverse conditions, however these treatments have some significant
drawbacks. It has also been demonstrated that nucleic acid
expression constructs that encode recombinant proteins are viable
solutions to the problems of frequent injections and high cost of
traditional recombinant therapy. By utilizing knowledge of specific
pituitary/hypothalamic pathways and the functionality of
extracranially secreted hormones, it is possible to treat many
conditions utilizing a plasmid-mediated introduction of a nucleic
acid construct into a subject. Furthermore, it has been shown that
some beneficial effects can be conferred to the offspring of female
subjects that have been treated utilizing recombinant proteins
during gestation and without treating the offspring directly. Thus,
this invention is related to the conferred beneficial effects in
offspring from GHRH treated mothers. More specifically this
invention discloses methods for altering pituitary development and
pituitary hormone secretion (e.g. prolactin) in the offspring from
female subjects treated with nucleic acid constructs that encode
GHRH.
SUMMARY
[0024] The present invention pertains to a plasmid-meditated gene
supplementation to alter pituitary development, and to increase
prolactin levels, in an offspring of a female subject. One
embodiment of the present invention pertains to administering to a
female subject a nucleic acid expression construct that encodes
growth hormone releasing hormone ("GHRH") to alter the pituitary
development and pituitary hormone secretion (e.g. prolactin, "PRL")
in the offspring from the female subject.
[0025] The present plasmid-mediated gene supplementation method
results in an increase in the pituitary lactotrophs (pituitary
cells that specifically produce prolactin), an increase in the
number and production of PRL by the pituitary gland, and an
increase in the prolactin levels in an offspring from the female
subject.
[0026] The female subject may be a mother, a female who has never
been pregnant or given birth before, or a surrogate mother, such as
impregnated by fetal transplantation. Although the nucleic acid
construct can be in a variety of different configurations, a
preferred embodiment of the construct comprises a promoter, a
nucleotide sequence, and a 3' untranslated region. The nucleic acid
sequence may comprise a growth hormone releasing hormone ("GHRH")
or a biological equivalent thereof, a myogenic promoter, and a
specified 3' untranslated region. Another embodiment includes the
use of modified GHRH analogs that have been engineered to be
protease resistant, but retain the functional biological activity
of the wild-type GHRH. The delivery of the nucleic acid expression
construct into the female subject may be accompanied or assisted.
Although electroporation is a preferred method to deliver the
nucleic acid expression construct into the cells of the female
subject, other approaches can be utilized for this purpose. In a
specific embodiment of the current invention, muscle cells are the
preferred cell type for delivery of the nucleic acid expression
construct, however, other cell types (e.g. somatic cells, stem
cells, or germ cells) can be utilized.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 shows the nucleic acid constructs that were used in
pregnant rats. Thirty micrograms of a pSP-HV-GHRH (SEQ ID#11)
nucleic acid construct was delivered into the tibialis anterior
muscle of rat dams at 16 days of gestation. Control dams were
injected with a similar construct driving the reporter,
beta-galactosidase. The injection was followed by in vivo
electroporation.
[0028] FIG. 2 shows the increased postnatal growth in offspring
from rats treated with the nucleic acid constructs pSP-HV-GHRH (SEQ
ID#11) and beta-galactosidase (".beta.-gal"). Significant weight
differences (*) for both sexes were recorded at 3 weeks of age
(p<0.05), and at 10 weeks of age (p<0.05). Female offspring
from .beta.-gal treated control dams ("CF"); female offspring of
pSP-HV-GHRH (SEQ ID#11)-treated dams--("IF"), male offspring from
.beta.-gal treated control dams ("CM"); male offspring of
pSP-HV-GHRH (SEQ ID#11)-treated dams--("IM"),
[0029] FIG. 3 shows muscle hypertrophy in the offspring of the
pSP-HV-GHRH (SEQ ID#11) treated dams. Both male and female
offspring from pSP-HV-GHRH (SEQ ID#11) treated animals had muscle
hypertrophy at 3 weeks of age. Gastrocnemius weight/body weight
("G/wt"); Tibialis anterior weight/body weight ("TA/wt"), wherein
the differences were significant at *=p<0.02;
#=p<0.008;.degree.=p<0.01. At 24 weeks of age the female
offspring of the pSP-HV-GHRH (SEQ ID#11) treated dams maintained
their muscle hypertrophy, whereas males were similar to controls.
Gastrocnemius weight/total body weight ("G/wt"); and Soleus
weight/total body weight ("S/wt") wherein the differences were
significant at .sctn.=p<0.007.
[0030] FIG. 4 shows the table and histogram of the fold activation
of IGF-I levels in offspring from pSP-HV-GHRH (SEQ ID#11)-treated
dams over the fold activation of the offspring from .beta.-gal
treated dams at 3, 12, and 24, weeks of age of the offspring. The
circulating IGF-I levels were measured by specific rat
radioimmunoassay ("RIA"). The histogram depicts fold IGF-I between
age and sex matched controls, wherein the values are significant
(*) at p<0.05. Female offspring from .beta.-gal treated control
dams ("CF"); female offspring of sp-HV-GHRH-treated dams--("IF"),
male offspring from .beta.-gal treated control dams ("CM"); male
offspring of sp-HV-GHRH-treated dams--("IM"). Rat IGF-I was
measured by specific radioimmunoassay (Diagnostic System
Laboratories, Webster, Tex.). The sensitivity of the assay was 0.8
ng/ml; intra-assay and inter-assay coefficients of variation were
2.4% and 4.1%, respectively.
[0031] FIG. 5 shows that both male and female offspring from
pSP-HV-GHRH (SEQ ID#11) treated dams had pituitary hypertrophy at 3
and 12 weeks, as measured by the pituitary weight/total body weight
ratio.
[0032] FIG. 6 shows a Northern blot analysis of pituitary tissue
from male offspring ("c3W") from .beta.-gal treated control dams
and male ("IM3W") and female ("IF3W") offspring from pSP-HV-GHRH
(SEQ ID#11) treated dams at 3 weeks. RNA was visualized using
probes for the 18s rRNA ("18S") loading marker; a rat growth
hormone releasing hormone specific cDNA probe ("GHRH"); a growth
hormone specific rat ("GH") cDNA probe; and a rat prolactin
specific cDNA probe. The intensity of the bands was determined
using a Phosphoimager (Molecular Dynamics) and associated software.
Histogram (B) shows fold increase in GH and PRL levels of the
offspring from the pSP-HV-GHRH (SEQ ID#11) treated dams over the GH
and PRL levels of the offspring from .beta.-gal treated control
dams. Pituitaries that had been snap frozen were homogenized in--I
will have to add the composition, and extracted. Total RNA was
DNase I treated and 20 .mu.g of RNA, DNA free was size separated in
1.5% agarose-formaldehyde gel and transferred to nylon membrane.
The membranes were hybridized with specific GHRH, GH (gift from Dr.
Kelly Mayo at Northwestern University, Chicago, Ill.) and PRL cDNA
riboprobes .sup.32P-labeled (gift from Dr. Kathleen Mahon at Baylor
College of Medicine, Houston, Tex.).
[0033] FIG. 7 shows immunostained sections of pituitary glands from
the 3 week old offspring of pSP-HV-GHRH (SEQ ID#11) and .beta.-gal
treated dams. Panel A depicts rat GH-specific staining and Panel B
depicts rat prolactin-specific staining, wherein the anterior
pituitary from offspring of .beta.-gal treated control dams ("CP");
anterior pituitary from offspring of pSP-HV-GHRH (SEQ ID#11)
treated dams ("TP"); and the anterior pituitary from offspring of
pSP-HV-GHRH (SEQ ID#11) treated dams with the immunostaining
wherein no primary antibody was added to the incubation reaction
("NC"), are shown.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Terms:
[0034] It will be readily apparent to one skilled in the art that
various substitutions and modifications may be made in the
invention disclosed herein without departing from the scope and
spirit of the invention.
[0035] The term "a" or "an" as used herein in the specification may
mean one or more. As used herein in the claim(s), when used in
conjunction with the word "comprising", the words "a" or "an" may
mean one or more than one. As used herein "another" may mean at
least a second or more.
[0036] The term "animal" as used herein refers to any species of
the animal kingdom. In preferred embodiments it refers more
specifically to humans, animals in their wild state, animals used
as pets (birds, dogs, cats, horses), animals used for work (horses,
cows, dogs) and animals which produce food (chickens, cows, fish),
farm animals (pigs, horses, cows, sheep, chickens) or are
themselves food (frogs, chickens, fish, crabs, lobsters, shrimp,
mussels, scallops, goats, boars, cows, lambs, pigs, ostrich, emu,
eel) and other animals well known to the art.
[0037] The term "effective amount" as used herein is defined as the
amount of the composition required to produce an effect in a host
which can be monitored using several endpoints known to those
skilled in the art. In a specific embodiment, these endpoints are
surrogate markers.
[0038] The term "feed conversion efficiency" as used herein is
defined as the amount of food an animal eats per day versus the
amount of weight gained by said animal. The terms "efficiency" or
"feed efficiency" as used herein is interchangeable with "feed
conversion efficiency."
[0039] The term "growth deficiencies" as used herein is defined as
any health status, medical condition or disease in which growth is
less than normal. The deficiency could be the result of an
aberration directly affecting a growth hormone pathway (such as the
GHRH-GH-IGF-I axis), indirectly affecting a growth hormone pathway,
or not affecting a growth hormone pathway at all.
[0040] The term "growth hormone" as used herein is defined as a
hormone which relates to growth and acts as a chemical messenger to
exert its action on a target cell.
[0041] The term "growth hormone releasing hormone" as used herein
is defined as a hormone which facilitates or stimulates release of
growth hormone.
[0042] The term "growth hormone releasing hormone analog" as used
herein is defined as protein which contains amino acid mutations
and/or deletions in the naturally occurring form of the amino acid
sequence (with no synthetic dextro or cyclic amino acids), but not
naturally occurring in the GHRH molecule, yet still retains its
function to enhance synthesis and secretion of growth hormone.
[0043] The term "growth hormone secretagogue receptor" (GHS-R) as
used herein is defined as a receptor for a small synthetic compound
which is associated, either directly or indirectly, with release of
growth hormone from the pituitary gland.
[0044] The term "ligand for a growth hormone secretagogue receptor"
as used herein is defined as any compound which acts as an agonist
on a growth hormone secretagogue receptor. The ligand may be
synthetic or naturally occurring. The ligand may be a peptide,
protein, sugar, carbohydrate, lipid, nucleic acid or a combination
thereof.
[0045] The term "myogenic" as used herein refers specifically to
muscle tissue.
[0046] The term "newborn" as used herein refers to an animal
immediately after birth and all subsequent stages of maturity or
growth.
[0047] The term "offspring" as used herein refers to a progeny of a
parent, wherein the progeny is an unborn fetus or a newborn.
[0048] The term "parenteral" as used herein refers to a mechanism
for introduction of material into an animal other than through the
intestinal canal. In specific embodiments, parenteral includes
subcutaneous, intramuscular, intravenous, intrathecal,
intraperitoneal, or
[0049] The term "pharmaceutically acceptable" as used herein refers
to a compound wherein administration of said compound can be
tolerated by a recipient mammal.
[0050] The term "secretagogue" as used herein refers to a natural
synthetic molecule that enhances synthesis and secretion of a
downstream-regulated molecule (e.g. GHRH is a secretagogue for
GH).
[0051] The term "somatotroph" as used herein refers to a cell which
produces growth hormone.
[0052] The term "lactotroph" as used herein refers to a cell which
produces prolactin.
[0053] The term "therapeutically effective amount" as used herein
refers to the amount of a compound administered wherein said amount
is physiologically significant. An agent is physiologically
significant if its presence results in technical change in the
physiology of a recipient animal. For example, in the treatment of
growth deficiencies, a composition which increases growth would be
therapeutically effective; in consumption diseases a composition
which would decrease the rate of loss or increase the growth would
be therapeutically effective.
[0054] The term "vector" as used herein refers to any vehicle which
delivers a nucleic acid into a cell or organism. Examples include
plasmids, viral vectors, liposomes, or cationic lipids. In a
specific embodiment, liposomes and cationic lipids are adjuvant
(carriers) that can be complexed with other vectors to increase the
uptake of plasmid or viral vectors by a target cell. In a preferred
embodiment, the vector comprises a promoter, a nucleotide sequence,
preferably encoding a growth hormone releasing hormone, its
biological equivalent, or its analog, and a 3' untranslated region.
In another preferred embodiment, the promoter, nucleotide sequence,
and 3' untranslated region are linked operably for expression in a
eukaryotic cell.
[0055] The term "nucleic acid expression construct" as used herein
refers to any type of genetic construct comprising a nucleic acid
coding for a RNA capable of being transcribed. The term "expression
vector" can also be used interchangeably.
[0056] The term "functional biological equivalent" of GHRH as used
herein is a polypeptide that has been engineered to contain a
distinct amino acid sequence while simultaneously having similar or
improved biologically activity when compared to the GHRH
polypeptide.
[0057] The term "functional nucleic acid equivalent" of a
referenced nucleic acid sequence as used herein means a nucleic
acid sequences that has been engineered to contain a distinct
nucleic acid sequences while simultaneously having similar or
improved functional activity when compared to the referenced
nucleic acid sequence. For example, because the universal code is
redundant, different codon sequences can express the same amino
acid (e.g. ACC, ACA and ACG all code for threonine). Thus, a codon
in an expression construct can be changed or optimized, but still
codes for an identical amino acid. Similarly, entire functional
nucleic acid sequences in an expression vector can be added or
deleted without changing the overall functionality of the
expression vector. For example, antibiotic resistant genes that are
used as selection markers for expression construct replication in
bacteria can be added, deleted, or interchanged without altering
the in vivo expression functionality the construct.
[0058] The term "subject" as used herein refers to any species of
the animal kingdom. In preferred embodiments it refers more
specifically to humans and animals used for: pets (e.g. cats, dogs,
etc.); work (e.g. horses, cows, etc.); food (chicken, fish, lambs,
pigs, etc); and all others known in the art.
[0059] The term "promoter" as used herein refers to a sequence of
DNA that directs the transcription of a gene. A promoter may be
"inducible", initiating transcription in response to an inducing
agent or, in contrast, a promoter may be "constitutive", whereby an
inducing agent does not regulate the rate of transcription. A
promoter may be regulated in a tissue-specific or tissue-preferred
manner, such that it is only active in transcribing the operable
linked coding region in a specific tissue type or types.
[0060] The term "coding region" as used herein refers to any
portion of the DNA sequence that is transcribed into messenger RNA
(mRNA) and then translated into a sequence of amino acids
characteristic of a specific polypeptide.
[0061] The term "analog" as used herein includes any mutant of
GHRH, or synthetic or naturally occurring peptide fragments of
GHRH, such as HV-GHRH (SEQ ID#1), TI-GHRH (SEQ ID#2), TV-GHRH (SEQ
ID#3), 15/27/28-GHRH (SEQ ID#4), (1-44)NH.sub.2 (SEQ ID#5) or
(1-40)OH (SEQ ID#6) forms, or any shorter form to no less than
(1-29) amino acids.
[0062] The term "delivery" as used herein is defined as a means of
introducing a material into a subject, a cell or any recipient, by
means of chemical or biological process, injection, mixing,
electroporation, sonoporation, or combination thereof, either under
or without pressure.
[0063] The term "growth hormone" ("GH") as used herein is defined
as a hormone that relates to growth and acts as a chemical
messenger to exert its action on a target cell.
[0064] The term "growth hormone releasing hormone" ("GHRH") as used
herein is defined as a hormone that facilitates or stimulates
release of growth hormone, and in a lesser extent other pituitary
hormones, as prolactin.
[0065] The term "regulator protein" as used herein refers protein
that increasing the rate of transcription in response to an
inducing agent.
[0066] The term "modified cells" as used herein is defined as the
cells from a subject that have an additional nucleic acid sequence
introduced into the cell.
[0067] The term "lean body mass" ("LBM") as used herein is defined
as the mass of the body of an animal attributed to non-fat tissue
such as muscle.
[0068] The term "cassette" as used herein is defined as one or more
transgene expression vectors.
[0069] The term "post-injection" as used herein refers to a time
period following the introduction of a nucleic acid cassette that
contains heterologous nucleic acid sequence encoding GHRH or
biological equivalent thereof into the cells of the subject and
allowing expression of the encoded gene to occur while the modified
cells are within the living organism.
[0070] The term "heterologous nucleic acid sequence" as used herein
is defined as a DNA sequence consisting of differing regulatory and
expression elements.
[0071] The term "regulator protein" as used herein refers to any
protein that can be used to control the expression of a gene.
[0072] The term "electroporation" as used herein refers to a method
that utilized electric pulses to deliver a nucleic acid sequence
into cells.
[0073] The term "poly-L-glutamate ("PLG")" as used herein refers to
a biodegradable polymer of L-glutamic acid that is suitable for use
as a vector or adjuvant for DNA transfer into cells with or without
electroporation.
[0074] In an embodiment of the present invention, a nucleic acid
expression construct is utilized in a plasmid meditated gene
supplementation method. The consequence of the claimed
supplementation method results in change in the pituitary lineage,
with increased number of lactotrophs and an increase in the
prolactin levels in an offspring from a female subject. The female
subject may be a mother, a female who has never been pregnant or
given birth before, or a surrogate mother, such as impregnated by
fetal transplantation. Modification of the pituitary gland lineage
in the female subject's offspring is achieved by utilizing a
nucleic acid expression construct that is delivered into the cells
of a female subject prior to or during gestation of the offspring.
Although the nucleic acid constructs comprise a variety of
different configurations, a preferred embodiment describes the
construct comprising a promoter, a nucleotide sequence, and a 3'
untranslated region. The nucleic acid sequence may comprise a
growth hormone releasing hormone ("GHRH") or biological equivalent
thereof, with a myogenic promoter, and a specified 3' untranslated
region. Further embodiments also include the use of modified
nucleic acid sequences that encode GHRH analogs that have been
engineered to be protease resistant, but retain the functional
biological activity of the wild-type GHRH.
[0075] Following the delivery of the nucleic acid expression
construct into the female subject, the process of electroporation
can be utilized to facilitate the uptake of the construct into the
cells of the female subject. Although electroporation is a
preferred method to deliver the nucleic acid expression construct
into the cells of the female subject, other approaches can be
utilized for this purpose, and are disclosed herein. In a specific
embodiment of the current invention, muscle cells are the preferred
cell type for delivery of the nucleic acid expression construct,
however, other cell types (e.g. somatic cells, stem cells, or germ
cells) can be utilized.
[0076] In order to assess growth effects of the growth hormone
releasing hormone ("GHRH") utilizing plasmid meditated gene
supplementation, several experiments that utilized myogenic vectors
with an encoded GHRH gene were conducted. The outcome resulted in a
co-pending patent application (i.e. U.S. patent application Ser.
No. 10/021,403 filed on Dec. 12, 2001, and is hereby incorporated
by reference) that disclosed methods used to treat pregnant sows in
the last trimester of gestation with a vector containing a nucleic
acid sequences for GHRH or biological equivalents thereof.
Injection of the nucleic acid expression construct was followed by
electroporation. Non-injected/electroporated sows were used as
controls. The piglets from the GHRH injected sow were found to be
bigger at birth. Cross-fostering studies were then performed, and
at weaning, the piglets from injected sows remained bigger than
controls. Cross-foster controls suckled on injected sows were also
significantly bigger than their littermates. Multiple biochemical
measurements were performed on the piglets and indicated that
piglets born to sows treated with plasmid meditated gene
supplementation of GHRH showed an increase in growth pattern over
normal levels. Although not wanting to be bound by theory, this
proof of principal experiment demonstrated that plasmid meditated
gene supplementation could be useful to enhance certain animal
characteristics throughout generations, while avoiding secondary
effects linked with classical supplementation treatments.
[0077] Although not wanting to be bound by theory, pituitary gland
development, including regulation and differentiation of
somatotrophs depends upon paracrine processes within the pituitary
itself and involves several growth factors and neuropeptides, such
as vasoactive intestinal peptide, angiotensin, endothelin, and
activin. Secretion of growth hormone ("GH") is stimulated by a
natural GH secretagogue called growth hormone releasing hormone
("GHRH"), and inhibited by somatostatin ("SS"), which are both
hypothalamic hormones. In healthy adult mammals, GH is released in
a highly regulated, distinctive pulsatile pattern, which occurs
when the stimulatory properties of GHRH are enabled by the
diminution or withdrawal of SS secretion. The episodic pattern of
GH secretion has profound importance for its biological activity
and is required for the induction of its physiological effects at
the peripheral level. Regulated GH secretion is essential for
optimal linear growth, homeostasis of carbohydrate, protein, and
fat metabolism, and for promoting a positive nitrogen balance
(Murray, et al., 2000). These effects are mediated largely by its
down-stream effector, insulin-like growth factor I ("IGF-I"). GH
secretion also is influenced in vivo by ghrelin, the newly
identified endogenous peptide ligand of the growth hormone
secretagogue receptor, and is dependent on GHRH (Hataya, et al.,
2001) for its GH-secretory activity (Horvath, et. al., 2001). In
this invention, we disclose a method to alter pituitary gland
development in the offspring of female subjects that were treated
with plasmid mediated gene supplementation of GHRH. This method
allows the pregnant subjects to be treated during the last
trimester of gestation and alter the lineage specification of the
pituitary gland as well as expression levels of growth hormone
("GH") and prolactin somatotrophs, without directly treating the
offspring.
[0078] Hypothalamic tissue-specific expression of the GHRH gene is
not required for its biological activity, as indicated by the
biological activity of extra-cranially secreted GHRH (Faglia, et.
al., 1992; Melmed, et. al., 1991). Recently, we showed that in
pigs, ectopic expression of a novel, serum protease-resistant
porcine GHRH driven by a synthetic muscle-specific promoter could
elicit robust GH and IGF-I responses following its in vivo
administration by intramuscular injection and electroporation
(Lopez-Calderon, et al., 1999). In the rat model, GHRH
administration is effective in inducing pituitary GH mRNA
expression and increasing GH content, as well as somatic growth,
with the endogenous episodic GHRH secretory pattern present in
males enhancing somatic growth over females (Borski, et. al.,
2000). Although, the intergenerational effects on the offspring of
pregnant animals with sustained GHRH expression are yet unknown,
studies in adult animals indicate a potential plasticity of the GH
somatotrophs in response to GHRH. Pathological GHRH stimulation
(irrespective of its source, from transgenic models to pancreatic
tumors) of GH secretion can result in proliferation, hyperplasia,
and adenomas of the adenohypophysial cells (Asa, et. al., 1992;
Sano, et. al., 1988). A preferred embodiment of the present
invention utilizes the growth hormone-releasing hormone analog
having a similar amino acid sequence of the wild-type ("wt")
plasmid. As used herein, the term wt or "wild-type" can be the
endogenous form of GHRH of any animal, or it may be a slightly
modified form of the hormone, such as the porcine GHRH. A skilled
artisan is aware that the endogenous GHRH has 44 amino acids, and
an amide group at the end, with the correct notation for that form
being (1-44)NH.sub.2-GHRH. In a specific embodiment, a form with
only 40 amino acids (lacking the last 4 amino acids) is used which
also does not contain an amide group, and may be referred to as
(1-40)OH-GHRH. This form as used herein may also be referred to as
wild-type because it does not contain internal mutations if
compared to the wild-type sequence, as opposed to other forms
discussed herein (such as the HV-GHRH discussed below) having
internal mutations introduced by site-directed mutagenesis. A
skilled artisan is aware that the 1-40 form and shorter forms (for
example, 1-32 or 1-29) exist naturally in humans and other mammals
(even in different types of GHRH secreting tumors), and they have
an activity comparable with the natural (1-44)NH.sub.2. In a
preferred embodiments of the present invention GHRH equivalents
with increased stability over wild type GHRH peptides are
utilized.
[0079] In other embodiments, different species of GHRH or an analog
of GHRH are within the scope of the invention. In an object of the
invention the residues encoded by the DNA are not modified
post-translationally, given the nature of the nucleic acid
administration.
[0080] The following species are within the scope of the present
invention. U.S. Pat. No. 4,223,019 discloses pentapeptides having
the amino acid sequence NH.sub.2--Y-Z-E-G-J-COOH, wherein Y is
selected from a group consisting of D-lysine and D-arginine; Z and
J are independently selected from a group consisting of tyrosine,
tryptophan, and phenylalanine; and E and G are independently
selected from a group consisting of D-tyrosine, D-tryptophan, and
D-phenylalanine. U.S. Pat. No. 4,223,020 discloses tetrapeptides
having the following amino acid sequence NH.sub.2-Y-Z-E-G-COOH
wherein Y and G are independently selected from a group consisting
of tyrosine, tryptophan, and phenylalanine; and Z and E are
independently selected from a group consisting of D-tyrosine,
D-tryptophan, and D-phenylalanine. U.S. Pat. No. 4,223,021
discloses pentapeptides having the following amino acid sequence
NH.sub.2-Y-Z-E-G-J-COOH wherein Y and G are independently selected
from a group consisting of tyrosine, tryptophan, and phenylalanine;
Z is selected from a group consisting of glycine, alanine, valine,
leucine, isoleucine, proline, hydroxyproline, serine, threonine,
cysteine, and methionine; and E and J are independently selected
from a group consisting of D-tyrosine, D-tryptophan, and
D-phenylalanine. U.S. Pat. No. 4,224,316 discloses novel
pentapeptides having the following amino acid sequence
NH.sub.2-Y-Z-E-G-J-COOH wherein Y and E are independently selected
from a group consisting of D-tyrosine, D-tryptophan, and
D-phenylalanine; Z and G are independently selected from a group
consisting of tyrosine, tryptophan, and phenylalanine; and J is
selected from a group consisting of glycine, alanine, valine,
leucine, isoleucine, proline, hydroxyproline, serine, threonine,
cysteine, methionine, aspartic acid, glutamic acid, asparagine,
glutamine, arginine, and lysine. U.S. Pat. No. 4,226,857 discloses
pentapeptides having the following amino acid sequence
NH.sub.2-Y-Z-E-G-J-COOH wherein Y and G are independently selected
from a group consisting of tyrosine, trytophan, and phenylalanine;
Z and J are independently selected from a group consisting of
D-tyrosine, D-tryptophan, and D-phenylalanine; and E is selected
from a group consisting of glycine, alanine, valine, leucine,
isoleucine, proline, hydroxyproline, serine, threonine, cysteine,
methionine, aspartic acid, glutamic acid, asparagine, glutamine,
and histidine. U.S. Pat. No. 4,228,155 discloses pentapeptides
having the following amino acid sequence NH.sub.2-Y-Z-E-G-J-COOH
wherein Y is selected from a group consisting of tyrosine,
D-tyrosine, tryptophan, D-tryptophan, phenylalanine, and
D-phenylalanine; Z and E are independently selected from a group
consisting of D-tyrosine, D-tryptophan, and D-phenylalanine; G is
selected from a group consisting of lysine and arginine; and J is
selected from a group consisting of glycine, alanine, valine,
leucine, isoleucine, proline, hydroxyproline, serine, threonine,
cysteine, and methionine. U.S. Pat. No. 4,228,156 discloses
tripeptides having the following amino acid sequence
NH.sub.2-Y-Z-E-COOH wherein Y and Z are independently selected from
a group consisting of D-tyrosine, D-tryptophan, and
D-phenylalanine; and E is selected from a group consisting of
tyrosine, tryptophan, and phenylalanine. U.S. Pat. No. 4,228,158
discloses pentapeptides having the following amino acid sequence
NH.sub.2-Y-Z-E-G-J-COOH wherein Y and G are independently selected
from a group consisting of tyrosine, tryptophan, and phenylalanine,
Z and E are independently selected from a group consisting of
D-tyrosine, D-tryptophan, and D-phenylalanine; and J is selected
from a group consisting of natural amino acids and the
D-configuration thereof. U.S. Pat. No. 4,833,166 discloses a
synthetic peptide having the formula:
H-Asp-Pro-Val-Asn-Ile-Arg-Ala-Phe-Asp-Asp-Val-Leu-Y wherein Y is OH
or NH.sub.2 or a non-toxic salt thereof and A synthetic peptide
having the formula:
H-Val-Glu-Pro-Gly-Ser-Leu-Phe-Leu-Val-Pro-Leu-Pro-Leu-Leu-Pro-Va-
l-His-Asp-Phe-Val-Gln-Gln-Phe-Ala-Gly-Ile-Y wherein Y is OH or
NH.sub.2 or a non-toxic salt thereof. Draghia-Akli, et al. (1997)
utilize a 228-bp fragment of hGHRH which encodes a 31-amino-acid
signal peptide and an entire mature peptide human GHRH(1-44)OH
(Tyr1 Leu44).
[0081] The embodiments of the present invention include: (1) a
method for changing the pituitary gland lineage, with an increased
number of somatotrophs and lactotrophs in an offspring; and (2) a
method for stimulating production of prolactin in an offspring at a
level greater than that associated with normal growth. All of these
methods include the step of introducing a nucleic acid construct or
plasmid vector into the mother of the offspring during gestation of
the offspring or during a previous pregnancy, wherein said vector
comprises a promoter; a nucleotide sequence, such as one encoding a
growth hormone releasing hormone or biological equivalent thereof;
and a 3' untranslated region operatively linked sequentially at
appropriate distances for functional expression.
[0082] It is an object of the present invention to change the
pituitary lineage, and increase levels of prolactin in an animal,
preferably an offspring from a mother. The preferred embodiments
allow modifications in the pituitary lineage, and increase levels
of prolactin in an animal for long terms, such as greater than a
few weeks or greater than a few months. In a specific embodiment,
this is achieved by administering growth hormone releasing hormone
into the mother of the offspring, preferably in a nucleic acid
form. In a preferred embodiment the GHRH nucleic acid is maintained
as an episome in a muscle cell. In a specific embodiment the
increase in GHRH affects the pituitary gland by increasing the
number of growth hormone producing cells, and consequently changes
their cellular lineage. Although not wanting to be bound by theory,
the ratio of somatotrophs (growth hormone producing cells) is
increased relative to other hormone producing cells in the
pituitary, such as corticotrophs, lactotrophs, gonadotrophs, etc.
Thus, the increase in growth hormone may be related to the rise in
the number of growth hormone-producing cells. Likewise, increases
in pituitary hormones, such as prolactin, may be related to the
rise in the number of prolactin producing cells in the
pituitary.
[0083] Prolactin is a single-chain protein hormone and is closely
related to growth hormone. It is chiefly secreted by lactotrophs in
the anterior pituitary. However, prolactin is also synthesized and
secreted by a broad range of other cells in the body, most
prominently various immune cells, the brain and the decidua of the
pregnant uterus. Prolactin is also found in the serum of normal
females and males. Prolactin secretion is pulsatile and also shows
diurnal variation, with the serum concentration increasing during
sleep and the lowest level occurs about 3 hours after waking. The
secretion of prolactin is increased by stress and appears to be
dependent upon a women's estrogen status.
[0084] The conventional view of prolactin is that the mammary gland
is its major target organ, and stimulating mammary gland
development along with milk production define its major functions.
Although these views are true, such descriptions fail to convey an
accurate depiction of this multifunctional hormone. For example, it
is difficult to find a mammalian tissue that does not express
prolactin receptors, and although the anterior pituitary is the
major source of prolactin, the hormone is synthesized and secreted
in many other tissues. Overall, several hundred different actions
have been reported for prolactin in various species. Some of
prolactin's major effects are summarized below.
[0085] Prolactin's major known functions are attributed with
mammary gland development, milk production and reproduction. In the
1920's it was found that extracts of the pituitary gland, when
injected into virgin rabbits, induced milk production. Subsequent
research demonstrated that prolactin has two major roles in milk
production: induction of lobuloalveolar growth of the mammary
gland; and stimulation of lactogenesis after birth. Prolactin,
along with cortisol and insulin, act together to stimulate
transcription of the genes that encode milk proteins. Prolactin is
also important in several non-lactational aspects of reproduction.
For example, in some species (e.g. rodents, dogs, skunks),
prolactin is necessary for maintenance of ovarian structures (i.e.
corpora lutea) that secrete progesterone. Mice that are homozygous
for an inactivated prolactin gene and thus incapable of secreting
prolactin are infertile due to defects in ovulation, fertilization,
preimplantation development and implantation. Prolactin also
appears to have stimulatory effects in some species on reproductive
or maternal behaviors such as nest building and retrieval of
scattered young.
[0086] Prolactin also appears to elicit effects in the immune
system. For example, the prolactin receptor is widely expressed by
immune cells, and some types of lymphocytes synthesize and secrete
prolactin. These observations suggest that prolactin may act as an
autocrine or paracrine modulator of immune activity. Conversely,
mice with homozygous deletions of the prolactin gene fail to show
significant abnormalities in immune responses. A considerable
amount of research is in progress to delineate the role of
prolactin in normal and pathologic immune responses. Although the
significance of these potential functions remains poorly
understood, it is clear that prolactin can stimulate and enhance
the immune system, which has been demonstrated in prior art (e.g.
U.S. Pat. Nos. 5,605,885; and 5,872,127). Furthermore, the present
invention indicates how increased prolactin levels are correlated
with increased IGF-I levels.
[0087] In a preferred embodiment the promoter is a synthetic
myogenic promoter and hGH 3' untranslated region (SEQ ID#8) is in
the 3' untranslated region. However, the 3 untranslated region may
be from any natural or synthetic gene. In a specific embodiment of
the present invention there is utilized a synthetic promoter,
termed SPc5-12 (SEQ ID#7) (Li, et al., 1999), which contains
proximal serum response elements ("SRE") from skeletal
.alpha.-actin, multiple MEF-2 sites, MEF-1 sites, and TEF-1 binding
sites, and greatly exceeds the transcriptional potencies of natural
myogenic promoters. In a preferred embodiment the promoter utilized
in the invention does not get shut off or reduced in activity
significantly by endogenous cellular machinery or factors. Other
elements, including trans-acting factor binding sites and enhancers
may be used in accordance with this embodiment of the invention. In
an alternative embodiment, a natural myogenic promoter is utilized,
and a skilled artisan is aware how to obtain such promoter
sequences from databases including the National Center for
Biotechnology Information (NCBI) GenBank database or the NCBI
PubMed site. A skilled artisan is aware that these World Wide Web
sites may be utilized to obtain sequences or relevant literature
related to the present invention.
[0088] In a specific embodiment the human growth hormone ("hGH")
hgH 3' (SEQ ID#8) untranslated region or polyadenylation signal is
utilized in a nucleic acid construct, such as a plasmid.
[0089] In specific embodiments the nucleic acid construct is
selected from the group consisting of a plasmid, a viral vector, a
liposome, or a cationic lipid. In further specific embodiments said
vector is introduced into myogenic cells or muscle tissue. In a
further specific embodiment said animal is a human, a pet animal, a
work animal, or a food animal.
[0090] In addition to the specific embodiment of introducing the
nucleic acid construct into the animal via a plasmid vector,
delivery systems for transfection of nucleic acids into the animal
or its cells known in the art may also be utilized. For example,
other non-viral or viral methods may be utilized. A skilled artisan
recognizes that a targeted system for non-viral forms of DNA or RNA
requires four components: 1) the DNA or RNA of interest; 2) a
moiety that recognizes and binds to a cell surface receptor or
antigen; 3) a DNA binding moiety; and 4) a lytic moiety that
enables the transport of the complex from the cell surface to the
cytoplasm. Further, liposomes and cationic lipids can be used to
deliver the therapeutic gene combinations to achieve the same
effect. Potential viral vectors include expression vectors derived
from viruses such as adenovirus, retrovirus, vaccinia virus, herpes
virus, and bovine papilloma virus. In addition, episomal vectors
may be employed. Other DNA vectors and transporter systems are
known in the art.
[0091] Vectors. One skilled in the art recognizes that expression
vectors derived from various bacterial plasmids, retroviruses,
adenovirus, herpes or from vaccinia viruses may be used for
delivery of nucleotide sequences to a targeted organ, tissue or
cell population. Methods which are well known to those skilled in
the art can be used to construct recombinant vectors that will
express the gene encoding the growth hormone releasing hormone
analog. Transient expression may last for a month or more with a
non-replicating vector and even longer if appropriate replication
elements are a part of the vector system, wherein the term "vector"
is used to refer to a carrier nucleic acid molecule into which a
nucleic acid sequence can be inserted for introduction into a cell
where the vector can be replicated and the nucleic acid sequence
can be expressed. The term vector can also be referred to as a
nucleic acid construct. A nucleic acid sequence can be "exogenous,"
which means that it is foreign to the cell into which the vector is
being introduced or that the sequence is homologous to a sequence
in the cell but in a position within the host cell nucleic acid in
which the sequence is ordinarily not found. Vectors include
plasmids, cosmids, viruses (bacteriophage, animal viruses, and
plant viruses), and artificial chromosomes (e.g., YACs). One of
skill in the art would be well equipped to construct a vector
through standard recombinant techniques, which are described in
Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated
herein by reference.
[0092] The term "expression vector" refers to a vector or nucleic
acid expression construct containing a nucleic acid sequence coding
for at least part of a gene product capable of being transcribed.
In a specific embodiment the nucleic acid sequence encodes part or
all of GHRH. In some cases, RNA molecules are then translated into
a protein, polypeptide, or peptide. In other cases, these sequences
are not translated, for example, in the production of antisense
molecules or ribozymes. Expression vectors can contain a variety of
"control sequences," which refer to nucleic acid sequences
necessary for the transcription and possibly translation of an
operably linked coding sequence in a particular host organism. In
addition to control sequences that govern transcription and
translation, vectors and expression vectors may contain nucleic
acid sequences that serve other functions as well and are described
infra.
[0093] In a preferred embodiment, the nucleic acid construction
construct or vector of the present invention is a plasmid which
comprises a synthetic myogenic (muscle-specific) promoter, a
nucleotide sequence encoding a growth hormone releasing hormone or
its analog, and a 3' untranslated region. In alternative
embodiments, the vectors is a viral vector, such as an
adeno-associated virus, an adenovirus, or a retrovirus. In
alternative embodiments, skeletal alpha-actin promoter, myosin
light chain promoter, cytomegalovirus promoter, or SV40 promoter
can be used. In other alternative embodiments, human growth
hormone, bovine growth hormone, SV40, or skeletal alpha actin 3'
untranslated regions are utilized in the vector.
[0094] Promoters and Enhancers. A "promoter" is a control sequence
that is a region of a nucleic acid sequence at which initiation and
rate of transcription are controlled. It may contain genetic
elements at which regulatory proteins and molecules may bind such
as RNA polymerase and other transcription factors. The phrases
"operatively positioned," "operatively linked," "under control,"
and "under transcriptional control" mean that a promoter is in a
correct functional location and/or orientation in relation to a
nucleic acid sequence to control transcriptional initiation and/or
expression of that sequence. A promoter may or may not be used in
conjunction with an "enhancer," which refers to a cis-acting
regulatory sequence involved in the transcriptional activation of a
nucleic acid sequence.
[0095] A promoter may be one of naturally-coding sequences located
upstream of the coding segment and/or exon. Such a promoter can be
referred to as "endogenous." Similarly, an enhancer may be one
naturally associated with a nucleic acid sequence, located either
downstream or upstream of that sequence. Alternatively, certain
advantages will be gained by positioning the coding nucleic acid
segment under the control of a recombinant or heterologous
promoter, which refers to a promoter that is not normally
associated with a nucleic acid sequence in its natural environment.
A recombinant or heterologous enhancer refers also to an enhancer
not normally associated with a nucleic acid sequence in its natural
environment. Such promoters or enhancers may include promoters or
enhancers of other genes, and promoters or enhancers isolated from
any other prokaryotic, viral, or eukaryotic cell, and promoters or
enhancers not "naturally occurring," i.e., containing different
elements of different transcriptional regulatory regions, and/or
mutations that alter expression. In addition to producing nucleic
acid sequences of promoters and enhancers synthetically, sequences
may be produced using recombinant cloning and/or nucleic acid
amplification technology, including PCR.TM.. Furthermore, it is
contemplated the control sequences that direct transcription and/or
expression of sequences within non-nuclear organelles such as
mitochondria, chloroplasts, and the like, can be employed as
well.
[0096] Naturally, it will be important to employ a promoter and/or
enhancer that effectively directs the expression of the DNA segment
in the cell type, organelle, and organism chosen for expression.
Those of skill in the art of molecular biology generally know the
use of promoters, enhancers, and cell type combinations for protein
expression. The promoters employed may be constitutive,
tissue-specific, inducible, and/or useful under the appropriate
conditions to direct high level expression of the introduced DNA
segment, such as is advantageous in the large-scale production of
recombinant proteins and/or peptides. The promoter may be
heterologous or endogenous. In a specific embodiment the promoter
is a synthetic myogenic promoter, such as is described in Li, et
al. (1999).
[0097] The identity of tissue-specific promoters or elements, as
well as assays to characterize their activity, is well known to
those of skill in the art. Examples of such regions include the
human LIMK2 gene, the somatostatin receptor 2 gene, murine
epididymal retinoic acid-binding gene, human CD4, mouse alpha2 (XI)
collagen, D1A dopamine receptor gene, insulin-like growth factor
II, human platelet endothelial cell adhesion molecule-1.
[0098] Initiation Signals and Internal Ribosome Binding Sites. A
specific initiation signal also may be required for efficient
translation of coding sequences. These signals include the ATG
initiation codon or adjacent sequences. Exogenous translational
control signals, including the ATG initiation codon, may need to be
provided. One of ordinary skill in the art would readily be capable
of determining this and providing the necessary signals. It is well
known that the initiation codon must be "in-frame" with the reading
frame of the desired coding sequence to ensure translation of the
entire insert. The exogenous translational control signals and
initiation codons can be either natural or synthetic. The
efficiency of expression may be enhanced by the inclusion of
appropriate transcription enhancer elements.
[0099] In certain embodiments of the invention, the use of internal
ribosome entry sites (IRES) elements are used to create multigene,
or polycistronic, messages. IRES elements are able to bypass the
ribosome scanning model of 5' methylated Cap dependent translation
and begin translation at internal sites. IRES elements from two
members of the picornavirus family (polio and encephalomyocarditis)
have been described, as well an IRES from a mammalian message. IRES
elements can be linked to heterologous open reading frames.
Multiple open reading frames can be transcribed together, each
separated by an IRES, creating polycistronic messages. By virtue of
the IRES element, each open reading frame is accessible to
ribosomes for efficient translation. Multiple genes can be
efficiently expressed using a single promoter/enhancer to
transcribe a single message.
[0100] Multiple Cloning Sites. Vectors can include a multiple
cloning site (MCS), which is a nucleic acid region that contains
multiple restriction enzyme sites, any of which can be used in
conjunction with standard recombinant technology to digest the
vector. "Restriction enzyme digestion" refers to catalytic cleavage
of a nucleic acid molecule with an enzyme that functions only at
specific locations in a nucleic acid molecule. Many of these
restriction enzymes are commercially available. Use of such enzymes
is widely understood by those of skill in the art. Frequently, a
vector is linearized or fragmented using a restriction enzyme that
cuts within the MCS to enable exogenous sequences to be ligated to
the vector. "Ligation" refers to the process of forming
phosphodiester bonds between two nucleic acid fragments, which may
or may not be contiguous with each other. Techniques involving
restriction enzymes and ligation reactions are well known to those
of skill in the art of recombinant technology.
[0101] Splicing Sites. Most transcribed eukaryotic RNA molecules
will undergo RNA splicing to remove introns from the primary
transcripts. Vectors containing genomic eukaryotic sequences may
require donor and/or acceptor splicing sites to ensure proper
processing of the transcript for protein expression.
[0102] Polyadenylation Signals. In expression, one will typically
include a polyadenylation signal to effect proper polyadenylation
of the transcript. The nature of the polyadenylation signal is not
believed to be crucial to the successful practice of the invention,
and/or any such sequence may be employed. Preferred embodiments
include the SV40 polyadenylation signal and/or the bovine or human
growth hormone polyadenylation signal, convenient and/or known to
function well in various target cells. Also contemplated as an
element of the expression cassette is a transcriptional termination
site. These elements can serve to enhance message levels and/or to
minimize read through from the cassette into other sequences.
[0103] Origins of Replication. In order to propagate a vector in a
host cell, it may contain one or more origins of replication sites
(often termed "ori"), which is a specific nucleic acid sequence at
which replication is initiated. Alternatively an autonomously
replicating sequence (ARS) can be employed if the host cell is
yeast.
[0104] Selectable and Screenable Markers. In certain embodiments of
the invention, the cells contain nucleic acid construct of the
present invention, a cell may be identified in vitro or in vivo by
including a marker in the expression vector. Such markers would
confer an identifiable change to the cell permitting easy
identification of cells containing the expression vector.
Generally, a selectable marker is one that confers a property that
allows for selection. A positive selectable marker is one in which
the presence of the marker allows for its selection, while a
negative selectable marker is one in which its presence prevents
its selection. An example of a positive selectable marker is a drug
resistance marker, such as the antibiotic resistance gene on the
plasmid constructs (such as ampicylin, gentamicin, tetracycline or
chloramphenicol).
[0105] Usually the inclusion of a drug selection marker aids in the
cloning and identification of transformants, for example, genes
that confer resistance to neomycin, puromycin, hygromycin, DHFR,
GPT, zeocin and histidinol are useful selectable markers. In
addition to markers conferring a phenotype that allows for the
discrimination of transformants based on the implementation of
conditions, other types of markers including screenable markers
such as GFP, whose basis is colorimetric analysis, are also
contemplated. Alternatively, screenable enzymes such as herpes
simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) may be utilized. One of skill in the art
would also know how to employ immunologic markers, possibly in
conjunction with FACS analysis. The marker used is not believed to
be important, so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable and screenable markers are well
known to one of skill in the art.
[0106] Host Cells. As used herein, the terms "cell," "cell line,"
and "cell culture" may be used interchangeably. All of these terms
also include their progeny, which is any and all subsequent
generations. It is understood that all progeny may not be identical
due to deliberate or inadvertent mutations. In the context of
expressing a heterologous nucleic acid sequence, "host cell" refers
to a prokaryotic or eukaryotic cell, and it includes any
transformable organism that is capable of replicating a vector
and/or expressing a heterologous gene encoded by a vector. A host
cell can, and has been, used as a recipient for vectors. A host
cell may be "transfected" or "transformed," which refers to a
process by which exogenous nucleic acid is transferred or
introduced into the host cell. A transformed cell includes the
primary subject cell and its progeny.
[0107] Host cells may be derived from prokaryotes or eukaryotes,
depending upon whether the desired result is replication of the
vector or expression of part or all of the vector-encoded nucleic
acid sequences. Numerous cell lines and cultures are available for
use as a host cell, and they can be obtained through the American
Type Culture Collection (ATCC), which is an organization that
serves as an archive for living cultures and genetic materials
(www.atcc.org). An appropriate host can be determined by one of
skill in the art based on the vector backbone and the desired
result. A plasmid or cosmid, for example, can be introduced into a
prokaryote host cell for replication of many vectors. Bacterial
cells used as host cells for vector replication and/or expression
include DH5a, JM109, and KC8, as well as a number of commercially
available bacterial hosts such as SURE.RTM. Competent Cells and
SOLOPACK.TM. Gold Cells (STRATAGENE.RTM., La Jolla). Alternatively,
bacterial cells such as E. coli LE392 could be used as host cells
for phage viruses.
[0108] Examples of eukaryotic host cells for replication and/or
expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO,
Saos, and PC 12. Many host cells from various cell types and
organisms are available and would be known to one of skill in the
art. Similarly, a viral vector may be used in conjunction with
either a eukaryotic or prokaryotic host cell, particularly one that
is permissive for replication or expression of the vector.
[0109] Some vectors may employ control sequences that allow it to
be replicated and/or expressed in both prokaryotic and eukaryotic
cells. One of skill in the art would further understand the
conditions under which to incubate all of the above described host
cells to maintain them and to permit replication of a vector. Also
understood and known are techniques and conditions that would allow
large-scale production of vectors, as well as production of the
nucleic acids encoded by vectors and their cognate polypeptides,
proteins, or peptides.
[0110] Expression Systems. Numerous expression systems exist that
comprise at least a part or all of the compositions discussed
above. Prokaryote- and/or eukaryote-based systems can be employed
for use with the present invention to produce nucleic acid
sequences, or their cognate polypeptides, proteins and peptides.
Many such systems are commercially and widely available.
[0111] The insect cell/baculovirus system can produce a high level
of protein expression of a heterologous nucleic acid segment, and
which can be bought, for example, under the name MAXBAC.RTM. 2.0
from INVITROGEN.RTM. and BACPACK.TM. BACULOVIRUS EXPRESSION SYSTEM
FROM CLONTECH.RTM..
[0112] Other examples of expression systems include
STRATAGENE.RTM.'s COMPLETE CONTROL.TM. Inducible Mammalian
Expression System, which involves a synthetic ecdysone-inducible
receptor, or its pET Expression System, an E. coli expression
system. Another example of an inducible expression system is
available from INVITROGEN.RTM., which carries the T-REX.TM.
(tetracycline-regulated expression) System, an inducible mammalian
expression system that uses the full-length CMV promoter.
INVITROGEN.RTM. also provides a yeast expression system called the
Pichia methanolica Expression System, which is designed for
high-level production of recombinant proteins in the methylotrophic
yeast Pichia methanolica. One of skill in the art would know how to
express a vector, such as an expression construct, to produce a
nucleic acid sequence or its cognate polypeptide, protein, or
peptide.
[0113] Mutagenesis. Where employed, mutagenesis will be
accomplished by a variety of standard, mutagenic procedures.
Mutation is the process whereby changes occur in the quantity or
structure of an organism. Mutation can involve modification of the
nucleotide sequence of a single gene, blocks of genes or whole
chromosome. Changes in single genes may be the consequence of point
mutations which involve the removal, addition or substitution of a
single nucleotide base within a DNA sequence, or they may be the
consequence of changes involving the insertion or deletion of large
numbers of nucleotides.
[0114] Mutations can arise spontaneously as a result of events such
as errors in the fidelity of DNA replication or the movement of
transposable genetic elements (transposons) within the genome. They
also are induced following exposure to chemical or physical
mutagens. Such mutation-inducing agents include ionizing
radiations, ultraviolet light and a diverse array of chemical such
as alkylating agents and polycyclic aromatic hydrocarbons all of
which are capable of interacting either directly or indirectly
(generally following some metabolic biotransformations) with
nucleic acids. The DNA lesions induced by such environmental agents
may lead to modifications of base sequence when the affected DNA is
replicated or repaired and thus to a mutation. Mutation also can be
site-directed through the use of particular targeting methods.
[0115] Site-Directed Mutagenesis. Structure-guided site-specific
mutagenesis represents a powerful tool for the dissection and
engineering of protein-ligand interactions. The technique provides
for the preparation and testing of sequence variants by introducing
one or more nucleotide sequence changes into a selected DNA.
[0116] Site-specific mutagenesis uses specific oligonucleotide
sequences which encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent, unmodified nucleotides. In
this way, a primer sequence is provided with sufficient size and
complexity to form a stable duplex on both sides of the deletion
junction being traversed. A primer of about 17 to 25 nucleotides in
length is preferred, with about 5 to 10 residues on both sides of
the junction of the sequence being altered.
[0117] The technique typically employs a bacteriophage vector that
exists in both a single-stranded and double-stranded form. Vectors
useful in site-directed mutagenesis include vectors such as the M13
phage. These phage vectors are commercially available and their use
is generally well known to those skilled in the art.
Double-stranded plasmids are also routinely employed in
site-directed mutagenesis, which eliminates the step of
transferring the gene of interest from a phage to a plasmid.
[0118] In general, one first obtains a single-stranded vector, or
melts two strands of a double-stranded vector, which includes
within its sequence a DNA sequence encoding the desired protein or
genetic element. An oligonucleotide primer bearing the desired
mutated sequence, synthetically prepared, is then annealed with the
single-stranded DNA preparation, taking into account the degree of
mismatch when selecting hybridization conditions. The hybridized
product is subjected to DNA polymerizing enzymes such as E. coli
polymerase I (Klenow fragment) in order to complete the synthesis
of the mutation-bearing strand. Thus, a heteroduplex is formed,
wherein one strand encodes the original non-mutated sequence, and
the second strand bears the desired mutation. This heteroduplex
vector is then used to transform appropriate host cells, such as E.
coli cells, and clones are selected that include recombinant
vectors bearing the mutated sequence arrangement. Comprehensive
information on the functional significance and information content
of a given residue of protein can best be obtained by saturation
mutagenesis in which all 19 amino acid substitutions are examined.
The shortcoming of this approach is that the logistics of
multi-residue saturation mutagenesis are daunting. Hundreds, and
possibly even thousands, of site specific mutants must be studied.
However, improved techniques make production and rapid screening of
mutants much more straightforward.
[0119] Dosage and Formulation. The composition (active ingredients;
herein, vectors comprising a promoter; a nucleotide sequence
encoding growth hormone releasing hormone ("GHRH") and a 3'
untranslated region operatively linked sequentially at appropriate
distances for functional expression) of this invention can be
formulated and administered to affect a variety of growth
deficiency states by any means that produces contact of the active
ingredient with the agent's site of action in the body of an
animal. The composition of the present invention is defined as a
vector containing a nucleotide sequence encoding the compound of
the invention, which is an amino acid sequence analog herein
described. Said composition is administered in sufficient quantity
to generate a therapeutically effective amount of said compound.
One skilled in the art recognizes that the terms "administered" and
"introduced" can be used interchangeably. They can be administered
by any conventional means available for use in conjunction with
pharmaceuticals, either as individual therapeutic active
ingredients or in a combination of therapeutic active ingredients.
In a preferred embodiment the active ingredient is administered
alone or in a buffer such as PBS, but may be administered with a
pharmaceutical carrier selected on the basis of the chosen route of
administration and standard pharmaceutical practice. Such
pharmaceutical compositions can be used for therapeutic or
diagnostic purposes in clinical medicine, both human and
veterinary. For example, they are useful in the treatment of
growth-related disorders such as hypopituitary dwarfism resulting
from abnormalities in growth hormone production. Furthermore they
can also be used to stimulate the growth or enhance feed conversion
efficiency of animals raised for meat production, to enhance milk
production, and stimulate egg production.
[0120] The dosage administered comprises a therapeutically
effective amount of active ingredient and will, of course, vary
depending upon known factors such as the pharmacodynamic
characteristics of the particular active ingredient and its mode
and route of administration; type of animal; age of the recipient;
sex of the recipient; reproductive status of the recipient; health
of the recipient; weight of the recipient; nature and extent of
symptoms; kind of concurrent treatment; frequency of treatment; and
the effect desired. Appropriate dosages of the vectors of the
invention to be administered will vary somewhat depending on the
individual subject and other parameters. The skilled practitioner
will be able to determine appropriate dosages based on the known
circulating levels of growth hormone associated with normal growth
and the growth hormone releasing activity of the vector. As is well
known in the art, treatment of a female or mother to produce bigger
animals will necessitate varying dosages from individual to
individual depending upon the degree of levels of increase of
growth hormone production required.
[0121] Thus, there is provided in accordance with this invention a
method of increasing growth of an offspring which comprises
administering to the female or mother of the offspring an amount of
the analog of this invention sufficient to increase the production
of growth hormone to levels greater than that which is associated
with normal growth. Normal levels of growth hormone vary
considerably among individuals and, for any given individual,
levels of circulating growth hormone vary considerably during the
course of a day.
[0122] Plasmid mediated gene supplementation and its vivo
expression. Recently, the delivery of specific genes to somatic
tissue in a manner that can correct inborn or acquired deficiencies
and imbalances was proved to be possible. Gene-based drug delivery
offers a number of advantages over the administration of
recombinant proteins. These advantages include the conservation of
native protein structure, improved biological activity, avoidance
of systemic toxicities, and avoidance of infectious and toxic
impurities. In addition, plasmid mediated gene supplementation
allows for prolonged exposure to the protein in the therapeutic
range, because the newly secreted protein is present continuously
in the blood circulation.
[0123] Although not wanting to be bound by theory, the primary
limitation of using recombinant protein is the limited availability
of protein after each administration. Plasmid mediated gene
supplementation using injectable DNA plasmid vectors overcomes
this, because a single injection into the subject's skeletal muscle
permits physiologic expression for extensive periods of time.
Injection of the vectors can promote the production of enzymes and
hormones in animals in a manner that more closely mimics the
natural process. Furthermore, among the non-viral techniques for
gene transfer in vivo, the direct injection of plasmid DNA into
muscle tissue is simple, inexpensive, and safe.
[0124] In a plasmid based expression system, a non-viral gene
vector may be composed of a synthetic gene delivery system in
addition to the nucleic acid encoding a therapeutic gene product.
In this way, the risks associated with the use of most viral
vectors can be avoided. The non-viral expression vector products
generally have low toxicity due to the use of "species-specific"
components for gene delivery, which minimizes the risks of
immunogenicity generally associated with viral vectors.
Additionally, no integration of plasmid sequences into host
chromosomes has been reported in vivo to date, so that this type of
gene transfer should neither activate oncogenes nor inactivate
tumor suppressor genes. As episomal systems residing outside the
chromosomes, plasmids have defined pharmacokinetics and elimination
profiles, leading to a finite duration of gene expression in target
tissues.
[0125] Efforts have been made to enhance the delivery of plasmid
DNA to cells by physical means including electroporation,
sonoporation, and pressure. Injection by electroporation involves
the application of a pulsed electric field to create transient
pores in the cellular membrane without causing permanent damage to
the cell, which allows for the introduction of exogenous molecules.
By adjusting the electrical pulse generated by an electroporetic
system, nucleic acid molecules can travel through passageways or
pores in the cell that are created during the procedure. The
electroporation technique has been used previously to transfect
tumor cells after injection of plasmid DNA, or to deliver the
antitumoral drug bleomycin to cutaneous and subcutaneous tumors.
Electroporation also has been used in rodents and other small
animals (Mir, et al., 1998; Muramatsu, et al., 1998). Advanced
techniques of intramuscular injections of plasmid DNA followed by
electroporation into skeletal muscle have been shown to lead to
high levels of circulating growth hormone releasing hormone
("GHRH") (Draghia-Akli, et al., 1999).
[0126] The ability of electroporation to enhance plasmid uptake
into the skeletal muscle has been well documented, as described
above. In addition, plasmid formulated with poly-L-glutamate
("PLG") or polyvinylpyrolidone (PVP) has been observed to increase
gene transfection and consequently expression to up to 10 fold into
mice, rats and dog muscle. Although not wanting to be bound by
theory, PLG will increase the transfection of the plasmid during
the electroporation process, not only by stabilizing the plasmid
DNA, and facilitating the intracellular transport through the
membrane pores, but also through an active mechanism. For example,
positively charged surface proteins on the cells could complex the
negatively charged PLG linked to plasmid DNA through
protein-protein interactions. When an electric field is applied,
the surface proteins reverse direction and actively internalize the
DNA molecules, which substantially increases the transfection
efficiency.
[0127] The use of directly injectable DNA plasmid vectors has been
limited in the past. The inefficient DNA uptake into muscle fibers
after simple direct injection has led to relatively low expression
levels, ant the duration of the transgene expression has been
short. The most successful previous clinical applications have been
confined to vaccines.
[0128] Although there are references in the art directed to
electroporation of eukaryotic cells with linear DNA, these examples
illustrate transfection into cell suspensions, cell cultures, and
the like, and the transfected cells are not present in a somatic
tissue.
[0129] Where appropriate, the plasmid mediated gene supplementation
vectors can be formulated into preparations in solid, semisolid,
liquid or gaseous forms in the ways known in the art for their
respective route of administration. Means known in the art can be
utilized to prevent release and absorption of the composition until
it reaches the target organ or to ensure timed-release of the
composition. A pharmaceutically acceptable form should be employed
which does not ineffectuate the compositions of the present
invention. In pharmaceutical dosage forms, the compositions can be
used alone or in appropriate association, as well as in
combination, with other pharmaceutically active compounds.
[0130] Accordingly, the pharmaceutical composition of the present
invention may be delivered via various routes and to various sites
in an animal body to achieve a particular effect (see, e.g.,
Rosenfeld et al. (1991); Rosenfeld et al., (1991a); Jaffe et al.,
1992). One skilled in the art will recognize that although more
than one route can be used for administration, a particular route
can provide a more immediate and more effective reaction than
another route. Local or systemic delivery can be accomplished by
administration comprising application or instillation of the
formulation into body cavities, inhalation or insufflation of an
aerosol, or by parenteral introduction, comprising intramuscular,
intravenous, peritoneal, subcutaneous, intradermal, as well as
topical administration.
[0131] One skilled in the art recognizes that different methods of
delivery may be utilized to administer a vector into a cell.
Examples include: (1) methods utilizing physical means, such as
electroporation (electricity), a gene gun (physical force) or
applying large volumes of a liquid (pressure); and (2) methods
wherein said vector is complexed to another entity, such as a
liposome or transporter molecule.
[0132] Accordingly, the present invention provides a method of
transferring a therapeutic gene to a host, which comprises
administering the vector of the present invention, preferably as
part of a composition, using any of the aforementioned routes of
administration or alternative routes known to those skilled in the
art and appropriate for a particular application. Effective gene
transfer of a vector to a host cell in accordance with the present
invention to a host cell can be monitored in terms of a therapeutic
effect (e.g. alleviation of some symptom associated with the
particular disease being treated) or, further, by evidence of the
transferred gene or expression of the gene within the host (e.g.,
using the polymerase chain reaction in conjunction with sequencing,
Northern or Southern hybridizations, or transcription assays to
detect the nucleic acid in host cells, or using immunoblot
analysis, antibody-mediated detection, mRNA or protein half-life
studies, or particularized assays to detect protein or polypeptide
encoded by the transferred nucleic acid, or impacted in level or
function due to such transfer).
[0133] These methods described herein are by no means
all-inclusive, and further methods to suit the specific application
will be apparent to the ordinary skilled artisan. Moreover, the
effective amount of the compositions can be further approximated
through analogy to compounds known to exert the desired effect.
[0134] Furthermore, the actual dose and schedule can vary depending
on whether the compositions are administered in combination with
other pharmaceutical compositions, or depending on interindividual
differences in pharmacokinetics, drug disposition, and metabolism.
Similarly, amounts can vary in in vitro applications depending on
the particular cell line utilized (e.g., based on the number of
vector receptors present on the cell surface, or the ability of the
particular vector employed for gene transfer to replicate in that
cell line). Furthermore, the amount of vector to be added per cell
will likely vary with the length and stability of the therapeutic
gene inserted in the vector, as well as the nature of the sequence,
and is particularly a parameter which needs to be determined
empirically, and can be altered due to factors not inherent to the
methods of the present invention (for instance, the cost associated
with synthesis). One skilled in the art can easily make any
necessary adjustments in accordance with the exigencies of the
particular situation.
[0135] It is an object of the present invention that a single
administration of a growth hormone releasing hormone is sufficient
for multiple gestation periods and also provides a therapy that
enhances the offspring's performances by enlarging the size of the
pituitary gland and increasing the levels of prolactin.
[0136] The invention may be better understood with reference to the
following examples, which are representative of some of the
embodiments of the invention, and are not intended to limit the
invention.
Example 1
GHRH Super-Active Analogs Increase GH Secretagogue Activity and
Stability
[0137] GHRH has a relatively short half-life of about 12 minutes in
the circulatory systems of both humans (Frohman et al., 1984) and
pigs. By employing GHRH analogs that prolong its biological
half-life and/or improve its GH secretagogue activity, enhanced GH
secretion is achieved. Plasmid vectors containing the muscle
specific synthetic promoter SPc5-12 (SEQ ID#7)were previously
described (Li, et al., 1999). Wild type and mutated procrine GHRH
cDNAs were generated by site directed mutagenesis of GHRH cDNA (SEQ
ID#9) (Altered Sites II in vitro Mutagenesis System, Promega,
Madison, Wis.), and cloned into the BamHI/Hind III sites of
pSPc5-12, to generate pSP-wt-GHRH (SEQ ID#15), or pSP-HV-GHRH (SEQ
ID#11), respectively. The 3' untranslated region (3'UTR) of growth
hormone was cloned downstream of GHRH cDNA. The resultant plasmids
contained mutated coding region for GHRH, and the resultant encoded
amino acid sequences were not naturally present in mammals. Several
different plasmids that encoded different mutated amino acid
sequences of GHRH or functional biological equivalent thereof are
as follows:
TABLE-US-00001 Plasmid Encoded Amino Acid Sequence wt-GHRH
YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNQEQGA-OH (SEQID#10) HV-GHRH
HVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SEQID#11) TI-GHRH
YIDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SEQID#12) TV-GHRH
YVDAIFTNSYRKVLAQLSARKLLQDTLNRQQGERNQEQGA-OH (SEQID#13)
15/27/28-GHRH YADAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH
(SEQID#14)
[0138] In general, the encoded GHRH or functional biological
equivalent thereof is of formula:
TABLE-US-00002 (SEQID#6)
-X.sub.1-X.sub.2-DAIFTNSYRKVL-X.sub.3-QLSARKLLQDI-X.sub.4-X.sub.5-RQQGERNQ-
EQGA-OH
wherein: X.sub.1 is a D- or L-isomer of an amino acid selected from
the group consisting of tyrosine ("Y"), or histidine ("H"); X.sub.2
is a D- or L-isomer of an amino acid selected from the group
consisting of alanine ("A"), valine ("V"), or isoleucine ("I");
X.sub.3 is a D- or L-isomer of an amino acid selected from the
group consisting of alanine ("A") or glycine ("G"); X.sub.4 is a D-
or L-isomer of an amino acid selected from the group consisting of
methionine ("M"), or leucine ("L"); X.sub.5 is a D- or L-isomer of
an amino acid selected from the group consisting of serine ("S") or
asparagines ("N").
[0139] Although not wanting to be bound by theory, the X.sub.3
position contains a Gly15 that was substituted for Ala15 to
increase .alpha.-helical conformation and amphiphilic structure to
decrease cleavage by trypsin-like enzymes (Su et al., 1991). GHRH
analogs with Ala15 substitutions display a 4-5 fold greater
affinity for the GHRH receptor (Campbell et al., 1991). To reduce
loss of biological activity due to oxidation of the Met, with
slightly more stable forms using molecules with a free
COOH-terminus (Kubiak et al., 1989), substitution of X.sub.4 and
X.sub.5, Met27 and Ser28 for Leu27 and Asn28 was performed. Thus, a
triple amino acid substitution mutant denoted as GHRH-15/27/28 was
formed. Dipeptidyl peptidase IV is the prime serum GHRH degradative
enzyme (Walter et al., 1980; Martin et al., 1993). The X.sub.1 and
X.sub.2 substitutions can be described as poorer dipeptidase
substrates were created by taking GHRH15/27/28 and then by
replacing Ile2 with Ala2 (GHRH-TI) or with Val2 (GHRH-TV), or by
converting Tyr1 and Ala2 for His1 and Val2.
[0140] In terms of "functional biological equivalents", it is well
understood by the skilled artisan that, inherent in the definition
of a "biologically functional equivalent" protein and/or
polynucleotide, is the concept that there is a limit to the number
of changes that may be made within a defined portion of the
molecule while retaining a molecule with an acceptable level of
equivalent biological activity. Functional biological equivalents
are thus defined herein as those proteins (and polynucleotides) in
selected amino acids (or codons) may be substituted. A peptide
comprising a functional biological equivalent of GHRH is a
polypeptide that has been engineered to contain distinct amino acid
sequences while simultaneously having similar or improved
biologically activity when compared to GHRH. For example one
biological activity of GHRH is to facilitate growth hormone ("GH")
secretion in the subject.
Example 2
DNA Constructs
[0141] In a specific embodiment, a plasmid of pSPc5-12-HV-GHRH is
utilized in the present invention. In another specific embodiment,
a plasmid vector is utilized wherein the plasmid comprises a
pVC0289 backbone; a promoter, such as of a GHRH cDNA, such as the
porcine HV-GHRH (the mutated HV-GHRH cDNA); and a 3' untranslated
region ("UTR"), such as from human growth hormone ("hGH").
[0142] To test the biological potency of the mutated porcine GHRH
cDNA sequences, plasmid vectors were engineered that were capable
of directing the highest level of skeletal muscle-specific gene
expression by a synthetic muscle promoter, SPc5-12, which contains
proximal serum response elements from skeletal .alpha.-actin
(SREs), multiple MEF-2 sites, multiple MEF-1 sites, and TEF-1
binding sites (Li, et al., 1999). A 228-bp fragment of porcine
GHRH, which encodes the 31 amino acid signal peptide and the entire
mature peptide porcine GHRH (Tyr1-Gly40) and or the GHRH mutants,
followed by the 3' untranslated region of human GH cDNA, were
incorporated into myogenic GHRH expression vectors by methods well
known in the art. The plasmid pSPc5-12 contains a 360 bp SacI/BamHI
fragment of the SPc5-12 synthetic promoter (Li, et al., 1999) in
the SacI/BamHI sites of pSK-GHRH backbone (Draghia-Akli et al.,
1997).
[0143] The wild type and mutated porcine GHRH cDNAs were obtained
by site directed mutagenesis of human GHRH cDNA utilizing the kit
Altered Sites II in vitro Mutagenesis System (Promega; Madison,
Wis.). The human GHRH cDNA was subcloned as a BamHI-Hind III
fragment into the corresponding sites of the pALTER Promega vector
and mutagenesis was performed according to the manufacturer's
directions. The porcine wild type cDNA was obtained from the human
cDNA by changing the human amino acids 34 and 38 using the primer
of nucleic acid sequence:
(5'-A-G-G-C-A-G-C-A-G-G-G-A-G-A-G-A-G-G-A-A-C-C-A-A-G-A-G-C-A-A-G-GA-G-C--
A-T-A-A-T-G-A-C-T-G-C-A-G-3'). The porcine HV mutations were made
with the primer of nucleic acid sequence:
(5'-A-C-C-C-T-C-A-G-G-A-T-G-C-G-G-C-G-G-C-A-C-G-T-A-G-A-T-G-C-C-A-T-C-T-T-
-C-A-C-C-A-A-C-3'). The porcine 15Ala mutation was made with the
primer of nucleic acid sequence:
(5'-C-G-G-A-A-G-G-T-G-C-T-G-G-C-C-C-A-G-C-T-G-T-C-C-G-C-C-3'). The
porcine 27Leu28Asn mutation was made with the primer of nucleic
acid sequence:
(5'-C-T-G-C-T-C-C-A-G-G-A-C-A-T-C-C-T-G-A-A-C-A-G-G-C-A-G-C-A-G-
-G-G-A-G-A-G-3'). Following mutagenesis the resulting clones were
sequenced to confirm correctness and subsequently subcloned into
the BamHI/Hind III sites of pSK-GHRH described in this Example by
methods well known to those in the art. Another plasmid that was
utilized included the pSP-SEAP construct that contains the
SacI/HindIII SPc5-12 fragment, SEAP gene and SV40 3'UTR from
pSEAP-2 Basic Vector (Clontech Laboratories, Inc., Palo Alto,
Calif.). Plasmids were grown in E. coli DH5.alpha. (Gibco BRL,
Carlsbad, Calif.). Endotoxin-free plasmid (Qiagen Inc., Chatsworth,
Calif., USA) preparations were diluted to 1 mg/ml in PBS, pH
7.4.
[0144] The plasmids described above do not contain polylinker,
IGF-I gene, a skeletal alpha-actin promoter or a skeletal alpha
actin 3' UTR/NCR. Furthermore, these plasmids were introduced by
muscle injection, followed by in vivo electroporation, as described
below.
Example 3
Intramuscular Injection of Plasmid and Electroporation
[0145] The nucleic acid constructs that were used in pregnant
female rats are shown in FIG. 1. Timed-pregnant Wistar female rats
were housed and cared for in the animal facility of Baylor College
of Medicine, Houston, Tex. Animals were maintained under
environmental conditions of 10 h light/14 h darkness, in accordance
with NIH Guide, USDA and Animal Welfare Act guidelines, and the
protocol was approved by the Institutional Animal Care and Use
Committee. The experiment was repeated twice. On day 16 of
gestation, dams (n=20/group) were weighed and anesthetized using a
combination of 42.8 mg/ml ketamine, 8.2 mg/ml xylazine and 0.7
mg/ml acepromazine, administered i.m. at a dose of 0.5-0.7 ml/kg.
The left tibialis anterior muscle was injected with 30 .mu.g of
pSP-HV-GHRH (SEQ ID#11) in 100 .mu.l PBS using 0.3 cc insulin
syringes (Becton-Dickinson, Franklin Lakes, N.J.). Control dams
were injected with a similar construct driving the reporter,
beta-galactosidase. For both groups, the injection was followed by
caliper electroporation, as described previously (Draghia-Akli, et
al. 1999). Briefly, two minutes after injection, the rat leg was
placed between a two needles electrode, 1 cm in length, 26 gauge, 1
cm between needles (Genetronics, San Diego, Calif.) and electric
pulses were applied. Three 60-ms pulses at a voltage of 100 V/cm
were applied in one orientation, then the electric field was
reversed, and three more pulses were applied in the opposite
direction. The pulses were generated with a T-820 Electro Square
Porator (Genetronics, San Diego, Calif.).
Example 4
Increased Body Weight for Offspring of Injected Dams
[0146] All injected dams gave birth at 20-22 days of gestation. In
the first study 240 offspring and in the second study 60 offspring
were analyzed from two weeks of age to 5 months of age (2, 3, 6, 8,
12, 16, 24 weeks after birth). Body weights were recorded at these
time points using the same calibrated balance. The average number
of pups per litter was similar between groups (pregnant rats
treated with GHRH ("I"), n=10.8.+-.0.75 pups/litter; controls ("C")
n=11.75.+-.0.5 pups/litter). At birth litter size was adjusted to
equalize the numbers of pups to 10 pups/dam.
[0147] At 2 weeks of age, the increased postnatal growth in
offspring from dams treated with the nucleic acid constructs
pSP-HV-GHRH (SEQ ID#11) ("I") and beta-galactosidase (".beta.-gal")
("C") were determined. Thus, at two weeks of age, the average pup
weight was 9% greater for the offspring of I dams compared to C
dams: I=31.47.+-.0.52 g/pup vs. C=28.86.+-.0.75 g/pup, p<0.014.
At 3 weeks of age, were determined and shown in FIG. 2. Body
weights for the female offspring of pSP-HV-GHRH (SEQ ID#11) treated
dams ("IF") was significantly increased (i.e. 51.97.+-.0.83 g) when
compared to the control females offspring ("CF") (47.07.+-.4.4 g,
p<0.043). Male offspring from pSP-HV-GHRH (SEQ ID#11) treated
dams ("IM") treated dams were 22% higher (i.e. 60.89.+-.1.02 g)
when compared to male offspring from control treated dams ("CM")
(i.e. 49.85.+-.4.9 g, p<0.001), as shown in FIG. 2. The weight
difference was maintained up to 10 weeks of age. However, at 24
weeks of age, the weight differences between IM and CM was not
significant. Significant weight differences (*) for both sexes were
recorded at 3 weeks of age (p<0.05), and at 10 weeks of age
(p<0.05). Female offspring from .beta.-gal treated control dams
("CF"); female offspring of sp-HV-GHRH-treated dams--("IF"), male
offspring from .beta.-gal treated control dams ("CM"); male
offspring of sp-HV-GHRH-treated dams--("IM").
[0148] The difference in weight between the progeny of treated and
untreated dams was maintained to adulthood. Although not wanting to
be bound by theory, this difference in weight was attributable
largely to enhanced growth of the musculature, which in the female
offspring was maintained for the entire period of the study (24
weeks). In male progeny, the higher muscle-to-body weight ratio was
maintained only to puberty. This gender difference might be
explained by differences in the hormonal profile of the two sexes.
Males and females have similar amounts of testosterone until
puberty, at which time testosterone levels increase much more
dramatically in males (Tipton, et al., 2001). Although not wanting
to be bound by theory, it is well-known that the postpubertal
gonadal steroid environment plays an important role in determining
anterior pituitary hormone synthesis and cellular composition. High
testosterone levels present in post-puberal rodent may blunt the
effect of increased GH production on the skeletal muscle. The rapid
increase in muscle mass in the postnatal "growing phase" is due to
growth of the muscle in both longitudinal and cross-sectional
dimensions. The remaining increase in muscle mass in the "steady
phase" (after the 10th week) is caused entirely by transverse
growth, depending mainly on the muscle fiber hypertrophy (but may
include increase of connective tissues) (Tamaki, et. al.,
1995).
Example 4
Increased Body/Muscle Weight for Offspring of Injected Dams
[0149] At the end of the experiment animals were anesthetized,
blood was collected by cardiac puncture, centrifuged immediately at
2.degree. C., and stored at -80.degree. C. prior to analysis.
Organs (heart, liver, spleen, kidney, pituitary, brain, adrenals,
skeletal muscles--tibialis anterior ("TA"), gastrocnemius ("G"),
soleus ("S"), and extensor digitorum longus ("EDL")) from the
offspring of treated and control dams were removed, weighed on an
analytical balance and snap frozen in liquid nitrogen. The tibia
was dissected and length was measured to the nearest 0.1 mm using
calipers. Organ weight/total body weight was similar in between T
and C at all time points after 3 weeks. At the first time point
tested (3 weeks) the liver weight/total body weight (TM
0.042.+-.0.0007 versus CM 0.035.+-.0.002, p<0.0004, and TF
0.0404.+-.0.0005 versus CF 0.0355.+-.0.0008, p<0.0002) and the
adrenal weight/total body weight (TM
4.4.times.10.sup.-4.+-.1.8.times.10.sup.-5, versus CM
3.6.times.10.sup.-4.+-.1.7.times.10.sup.-5, p<0.03, and TF
4.3.times.10.sup.4.+-.0.9.times.10.sup.-5, versus CF
3.2.times.10.sup.-4.+-.3.5.times.10.sup.-5, p<0.0003) were
increased in the offspring of the T dams. No differences in between
organ weights/total body weights were noticed at subsequent time
points analyzed. No associated pathology was observed in any of the
animals through the entire period of the study.
[0150] In contrast, both male and female offspring from pSP-HV-GHRH
(SEQ ID#11) treated dams had muscle hypertrophy at 3 weeks of age
with 10-12% differences in the gastrocnemius ("G") and tibialis
anterior ("TA") muscle weights, even after the differences in body
weights were adjusted. Gastrocnemius weight/body weight ("G/wt");
Tibialis anterior weight/body weight ("TA/wt"), wherein the
differences were significant at *=p<0.02;
#=p<0.008;.degree.=p<0.01, as shown in FIG. 3. At 24 weeks of
age the female offspring IF of the pSP-HV-GHRH (SEQ ID#11) treated
dams maintained their muscle hypertrophy, whereas males IM were
similar to controls.
Example 5
Increased Serum IGF-I Levels for Offspring of Injected Dams
[0151] An indication of increased systemic levels of GHRH and GH is
an increase in serum IGF-I concentration. Serum rat IGF-I was
significantly higher in offspring of T dams compared to those from
C dams at all time points tested until 24 weeks. FIG. 4 shows the
table and histogram of the fold activation of IGF-I levels in
offspring from sp-HV-GHRH-treated dams over the fold activation of
the offspring from .beta.-gal treated dams at 3, 12, and 24, weeks
of age of the offspring. The circulating IGF-I levels were measured
by specific rat radioimmunoassay ("RIA"). The histogram depicts
fold IGF-I between age and sex matched controls, wherein the values
are significant (*) at p<0.05. Female offspring from .beta.-gal
treated control dams ("CF"); female offspring of sp-HV-GHRH-treated
dams--("IF"), male offspring from .beta.-gal treated control dams
("CM"); male offspring of sp-HV-GHRH-treated dams--("IM"). Although
not wanting to be bound by theory, the normal mechanisms
responsible for the increase in serum GH levels that occur during
pregnancy include: an increase in GH gene expression in the
pituitary, a decrease in somatostatin secretion from the
hypothalamus, an increase in immunoreactive-IGF-I content in both
the hypothalamus and in the pituitary, and a significant decrease
in circulating IGF-I. This state of GH resistance with a higher
GH/IGF-I ratio could be important in providing supplementary
nutrients to the fetus during the latter part of gestation when
fetal growth is most rapid (Escalada et al., 1997). Our therapy
further stimulated the maternal GHRH axis, fact that may explain
the increased weight of the offspring of the treated animals at two
weeks of age. Although not wanting to be bound by theory, it is
also postulated that a ghrelin gene expression in the pituitary is
developmentally regulated, and its expression is increased
following GHRH infusion; the pituitary ghrelin/GHS-R signaling
system could modulate the regulation of GH secretion by GHRH
(Kamegai, et. al., 2001). Although not wanting to be bound by
theory, some other possible explanations include: increased
placental transport of nutrients. Postnatal, the growth curve could
also be changed by increased milk production in the dam. Milk and
colostrums contain a variety of proteins, peptides and steroids
that possess biological activity (Grosvenor, et. al., 1993), that
can be absorbed in the early neonatal period (before the "gut
closure") into serum as intact and/or low-molecular weight
processed forms (Gonnella, et. al., 1989). It is known that is
rats, the concentration of GHRH in milk exceeds that in plasma by
several fold; in addition the neonatal rat pituitary exhibits a
greater sensitivity to the stimulatory effects of GHRH (Szabo, et.
al., 1986). Thus, milk GHRH may function transiently to stimulate
pituitary differentiation of the offspring.
Example 6
Increased Serum IGF-I Levels for Offspring of Injected Dams
[0152] As shown in FIG. 5, both male and female offspring from
pSP-HV-GHRH (SEQ ID#11) treated dams had pituitary hypertrophy at 3
and 12 weeks. The pituitary glands were dissected and weighed
within the first few minutes post-mortem. Pituitary weight adjusted
for body weight was significantly increased at least to 12 weeks of
age; this difference was more prominent for IF. Although not
wanting to be bound by theory, the increase in pituitary weight was
probably due to hyperplasia of the somatotrophs and
lactotrophs.
[0153] The hypothesis that GHRH has a specific hypertrophic effect
on GH and prolactin secreting cells is supported by the mRNA levels
and immunohistochemical experimental evidence. For example, FIG. 6A
shows a Northern blot analysis of pituitary tissue from male
offspring ("c3W") from .beta.-gal treated control dams and male
("IM3W") and female ("IF3W") offspring from pSP-HV-GHRH (SEQ ID#11)
treated dams at 3 weeks. RNA was visualized using probes for the
18s rRNA ("18S") loading marker; a rat growth hormone releasing
hormone specific cDNA probe ("GHRH"); a growth hormone specific rat
("GH") cDNA probe; and a rat prolactin specific cDNA probe. The
intensity of the bands was determined using a Phosphoimager
(Molecular Dynamics) and associated software. Histogram (6B) shows
2.5-fold increase in GH and PRL levels of the offspring from the
pSP-HV-GHRH (SEQ ID#11) treated dams over the GH and PRL levels of
the offspring from .beta.-gal treated control dams. This difference
in response was associated with a diminution of 20% in the
endogenous rat GHRH mRNA levels.
[0154] Sections of pituitary glands were fixed immediately after
dissection in 3% paraformaldehyde in PBS overnight. After fixation,
samples were washed and stored in 70% ethanol until analyzed.
Pituitary glands were paraffin embedded, and five micron-thick
sections were cut, deparaffinized, and washed in PBS. Sections were
blocked using a solution of 5% normal goat serum, 1% BSA, 0.05%
Tween 20 in PBS for 1 hour at room temperature. The sections then
were incubated for 2 hours at room temperature with the primary
antibodies, rabbit-antirat-growth hormone (AFP5672099Rb, National
Hormone and Peptide Program--NHPP) and rabbit-antirat-prolactin
(AFP425-10-91 (NHPP)) diluted 1:2000 and 1:10000, respectively.
After, washing off the primary antibodies, secondary
peroxidase-coupled goat anti-rabbit IgG antibody (Sigma) at 1:5000
dilution was subsequently applied for 30 minutes at room
temperature. Slides were washed in distilled water in between every
step of the procedure. Peroxidase activity was revealed using a DAB
substrate for 4 minutes (Vector laboratories, Burlingame, Calif.).
Slides were counterstained with hematoxylin to visualize cell
morphology and nuclei. Digital images of the slides were captured
using a CoolSnap digital color camera (Roper Scientific, Tucson,
Ariz.) with MetaMorph software (Universal Imaging Corporation,
Downington, Pa.) and a Zeiss Axioplan 2 microscope with a
(.times.40) objective (numerical aperture 0.75 plan).
[0155] At the same age, pituitary sections immuno-stained with a
rat GH-specific antibody (FIG. 7A), depicted an increased number of
GH-immunoreactive cells (76% versus controls 39%), with an
increased amount of GH per immunoreactive somatotroph. For each
animal immunoreactive cells/total number of cells was counted in at
least 5 fields and averaged. Similarly, sections stained with a rat
prolactin specific antibody (FIG. 7B), showed an increase in the
number of prolactin-producing cells (25% versus 9% in
controls).
[0156] In contrast to our results, previous studies conducted in
GHRH transgenic animals observed a certain developmental pattern,
with pituitary weight increasing mainly after the first 6 months of
life, and with 70% of the glands contained grossly visible
adenomas, that stained positively for GH, whereas only some showed
scattered PRL staining. Although not wanting to be bound by theory,
in our methods, rat dams were treated in the last trimester of
gestation and pups pituitaries were most probably exposed to the
hormone only a limited period of time, which determined a change in
pituitary cell lineage, with somatotroph and lactotroph
hyperplasia, without neoplastic changes within the pituitary.
[0157] In summary of the prior examples, enhanced animal growth
occurred in offspring following a single electroporated injection
of a plasmid expressing a mutated growth hormone releasing hormone
(GHRH) cDNA, into the tibialis anterior muscles of pregnant female
subjects. The newborn offspring from treated females were
significantly bigger at birth. The longitudinal weight and body
composition studies showed a difference in between the two sexes
and with age. The offspring from treated females showed plasma
IGF-I levels that were significantly elevated over offspring from
control treated female subjects. The offspring from treated females
from had larger pituitary glands, with apparent somatotroph
hyperplasia and increased levels of pituitary derived hormones
(e.g. GH and prolactin).
[0158] The use of recombinant GHRH, which is an upstream stimulator
of GH, may be an alternate strategy to increase the size of the
pituitary gland and prolactin levels in the offspring of treated
mothers. However, the high cost of the recombinant peptides and the
required frequency of administration currently limit the widespread
use of such a recombinant treatment. These major drawbacks can be
obviated by using a plasmid meditated gene supplementation method
to direct the ectopic production of GHRH in pregnant females.
Although not wanting to be bound by theory, similar treatments with
recombinant GH or prolactin during the immediate postnatal period
of the offspring will specifically increase pituitary size,
increase prolactin levels, mitigate the deposition of body fat in
later life, whilst enhancing lean tissue deposition, and enhancing
the immune function.
[0159] By utilizing knowledge of specific pituitary/hypothalamic
pathways and the functionality of extracranially secreted hormones,
it is possible to treat many conditions utilizing a plasmid
mediated introduction of a nucleic acid construct into a subject.
Furthermore, it has been shown that some beneficial effects can be
conferred to the offspring of female subjects that have been
treated utilizing a plasmid mediated introduction of a nucleic acid
construct, without treating the offspring directly. The consequence
of the claimed supplementation method results is modification in
the pituitary lineage, with an increased number of somatotrophs and
lactotrophs and an increase in the prolactin levels in an offspring
from the female subject. The female subject may be a mother, a
female who has never been pregnant or given birth before, or a
surrogate mother, such as impregnated by fetal transplantation. The
benefit of this invention shows that offspring from animals that
have been treated with a plasmid meditated gene supplementation
method would benefit indirectly from the therapy without being
individually treated. Such a method, would save a considerable
amount financial resources for treating subjects, and if only
mothers were treated, a reduction in the time for implementing such
a therapy would also be expected.
[0160] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objectives and obtain
the ends and advantages mentioned as well as those inherent
therein. Growth hormone, growth hormone releasing hormone, analogs,
plasmids, vectors, pharmaceutical compositions, treatments,
methods, procedures and techniques described herein are presently
representative of the preferred embodiments and are intended to be
exemplary and are not intended as limitations of the scope. Changes
therein and other uses will occur to those skilled in the art which
are encompassed within the spirit of the invention or defined by
the scope of the pending claims.
Sequence CWU 1
1
15140PRTartificial sequenceThis is a growth hormone releasing
hormone ("GHRH") analog. 1His Val Asp Ala Ile Phe Thr Asn Ser Tyr
Arg Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp
Ile Leu Asn Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly
Ala35 40240PRTartificial sequenceThis is a growth hormone releasing
hormone ("GHRH") analog. 2Tyr Ile Asp Ala Ile Phe Thr Asn Ser Tyr
Arg Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp
Ile Leu Asn Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly
Ala35 40340PRTartificial sequenceThis is a growth hormone releasing
hormone ("GHRH") analog. 3Tyr Val Asp Ala Ile Phe Thr Asn Ser Tyr
Arg Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp
Ile Leu Asn Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly
Ala35 40440PRTartificial sequenceThis is a growth hormone releasing
hormone ("GHRH") analog. 4Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr
Arg Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp
Ile Leu Asn Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly
Ala35 40544PRTartificial sequenceThis is a growth hormone releasing
hormone ("GHRH") analog. 5Thr Ala Asp Ala Ile Phe Thr Asn Ser Tyr
Arg Lys Val Leu Gly Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp
Ile Met Ser Arg Gln Gln Gly20 25 30Glu Ser Asn Gln Glu Arg Gly Ala
Arg Ala Arg Leu35 40640PRTartificial sequenceThis is the synthetic
sequence for GHRH (1-40)OH. 6Xaa Xaa Asp Ala Ile Phe Thr Asn Ser
Tyr Arg Lys Val Leu Xaa Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln
Asp Ile Xaa Xaa Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly
Ala35 407323DNAartificial sequenceThis is a nucleic acid sequence
of a eukaryotic promoter c5-12. 7cggccgtccg ccctcggcac catcctcacg
acacccaaat atggcgacgg gtgaggaatg 60gtggggagtt atttttagag cggtgaggaa
ggtgggcagg cagcaggtgt tggcgctcta 120aaaataactc ccgggagtta
tttttagagc ggaggaatgg tggacaccca aatatggcga 180cggttcctca
cccgtcgcca tatttgggtg tccgccctcg gccggggccg cattcctggg
240ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg
cggcccacga 300gctacccgga ggagcgggag gcg 3238190DNAartificial
sequenceNucleic acid sequence of a human growth hormone poly A
tail. 8gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt
tgccactcca 60gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
ctaggtgtcc 120ttctataata ttatggggtg gaggggggtg gtatggagca
aggggcaagt tgggaagaca 180acctgtaggg 1909219DNAartificial
sequencecDNA for Porcine growth hormone releasing hormone.
9atggtgctct gggtgttctt ctttgtgatc ctcaccctca gcaacagctc ccactgctcc
60ccacctcccc ctttgaccct caggatgcgg cggcacgtag atgccatctt caccaacagc
120taccggaagg tgctggccca gctgtccgcc cgcaagctgc tccaggacat
cctgaacagg 180cagcagggag agaggaacca agagcaagga gcataatga
2191040PRTartificial sequenceAmino acid sequence for porcine growth
hormone releasing hormone. 10Tyr Ala Asp Ala Ile Phe Thr Asn Ser
Tyr Arg Lys Val Leu Gly Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln
Asp Ile Met Ser Arg Gln Gln Gly20 25 30Glu Arg Asn Gln Glu Gln Gly
Ala35 40113534DNAartificial sequenceNucleic acid sequence of the
HV-GHRH plasmid. 11gttgtaaaac gacggccagt gaattgtaat acgactcact
atagggcgaa ttggagctcc 60accgcggtgg cggccgtccg ccctcggcac catcctcacg
acacccaaat atggcgacgg 120gtgaggaatg gtggggagtt atttttagag
cggtgaggaa ggtgggcagg cagcaggtgt 180tggcgctcta aaaataactc
ccgggagtta tttttagagc ggaggaatgg tggacaccca 240aatatggcga
cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg
300cattcctggg ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg
gggccggcgg 360cggcccacga gctacccgga ggagcgggag gcgccaagct
ctagaactag tggatcccaa 420ggcccaactc cccgaaccac tcagggtcct
gtggacagct cacctagctg ccatggtgct 480ctgggtgttc ttctttgtga
tcctcaccct cagcaacagc tcccactgct ccccacctcc 540ccctttgacc
ctcaggatgc ggcggcacgt agatgccatc ttcaccaaca gctaccggaa
600ggtgctggcc cagctgtccg cccgcaagct gctccaggac atcctgaaca
ggcagcaggg 660agagaggaac caagagcaag gagcataatg actgcaggaa
ttcgatatca agcttatcgg 720ggtggcatcc ctgtgacccc tccccagtgc
ctctcctggc cctggaagtt gccactccag 780tgcccaccag ccttgtccta
ataaaattaa gttgcatcat tttgtctgac taggtgtcct 840tctataatat
tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa
900cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
caatcttggc 960tcactgcaat ctccgcctcc tgggttcaag cgattctcct
gcctcagcct cccgagttgt 1020tgggattcca ggcatgcatg accaggctca
gctaattttt gtttttttgg tagagacggg 1080gtttcaccat attggccagg
ctggtctcca actcctaatc tcaggtgatc tacccacctt 1140ggcctcccaa
attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga
1200ttttaaaata actataccag caggaggacg tccagacaca gcataggcta
cctggccatg 1260cccaaccggt gggacatttg agttgcttgc ttggcactgt
cctctcatgc gttgggtcca 1320ctcagtagat gcctgttgaa ttcgataccg
tcgacctcga gggggggccc ggtaccagct 1380tttgttccct ttagtgaggg
ttaatttcga gcttggcgta atcatggtca tagctgtttc 1440ctgtgtgaaa
ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
1500gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
cgctcactgc 1560ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
atgaatcggc caacgcgcgg 1620ggagaggcgg tttgcgtatt gggcgctctt
ccgcttcctc gctcactgac tcgctgcgct 1680cggtcgttcg gctgcggcga
gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1740cagaatcagg
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
1800accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
gacgagcatc 1860acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
aggactataa agataccagg 1920cgtttccccc tggaagctcc ctcgtgcgct
ctcctgttcc gaccctgccg cttaccggat 1980acctgtccgc ctttctccct
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2040atctcagttc
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
2100agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
gtaagacacg 2160acttatcgcc actggcagca gccactggta acaggattag
cagagcgagg tatgtaggcg 2220gtgctacaga gttcttgaag tggtggccta
actacggcta cactagaaga acagtatttg 2280gtatctgcgc tctgctgaag
ccagttacct tcggaaaaag agttggtagc tcttgatccg 2340gcaaacaaac
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
2400gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
gctcagaaga 2460actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc
gggagcggcg ataccgtaaa 2520gcacgaggaa gcggtcagcc cattcgccgc
caagctcttc agcaatatca cgggtagcca 2580acgctatgtc ctgatagcgg
tccgccacac ccagccggcc acagtcgatg aatccagaaa 2640agcggccatt
ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
2700cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc
gcgagcccct 2760gatgctcttc gtccagatca tcctgatcga caagaccggc
ttccatccga gtacgtgctc 2820gctcgatgcg atgtttcgct tggtggtcga
atgggcaggt agccggatca agcgtatgca 2880gccgccgcat tgcatcagcc
atgatggata ctttctcggc aggagcaagg tgagatgaca 2940ggagatcctg
ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
3000cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
cgcgctgcct 3060cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt
gacaaaaaga accgggcgcc 3120cctgcgctga cagccggaac acggcggcat
cagagcagcc gattgtctgt tgtgcccagt 3180catagccgaa tagcctctcc
acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 3240caatcatgcg
aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc
3300agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca
accttaccag 3360agggcgcccc agctggcaat tccggttcgc ttgctgtcca
taaaaccgcc cagtctagca 3420actgttggga agggcgatcg gtgcgggcct
cttcgctatt acgccagctg gcgaaagggg 3480gatgtgctgc aaggcgatta
agttgggtaa cgccagggtt ttcccagtca cgac 3534123534DNAartificial
sequenceNucleic acid sequence of the TI-GHRH plasmid. 12gttgtaaaac
gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcc 60accgcggtgg
cggccgtccg ccctcggcac catcctcacg acacccaaat atggcgacgg
120gtgaggaatg gtggggagtt atttttagag cggtgaggaa ggtgggcagg
cagcaggtgt 180tggcgctcta aaaataactc ccgggagtta tttttagagc
ggaggaatgg tggacaccca 240aatatggcga cggttcctca cccgtcgcca
tatttgggtg tccgccctcg gccggggccg 300cattcctggg ggccgggcgg
tgctcccgcc cgcctcgata aaaggctccg gggccggcgg 360cggcccacga
gctacccgga ggagcgggag gcgccaagct ctagaactag tggatcccaa
420ggcccaactc cccgaaccac tcagggtcct gtggacagct cacctagctg
ccatggtgct 480ctgggtgttc ttctttgtga tcctcaccct cagcaacagc
tcccactgct ccccacctcc 540ccctttgacc ctcaggatgc ggcggtatat
cgatgccatc ttcaccaaca gctaccggaa 600ggtgctggcc cagctgtccg
cccgcaagct gctccaggac atcctgaaca ggcagcaggg 660agagaggaac
caagagcaag gagcataatg actgcaggaa ttcgatatca agcttatcgg
720ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
gccactccag 780tgcccaccag ccttgtccta ataaaattaa gttgcatcat
tttgtctgac taggtgtcct 840tctataatat tatggggtgg aggggggtgg
tatggagcaa ggggcaagtt gggaagacaa 900cctgtagggc ctgcggggtc
tattgggaac caagctggag tgcagtggca caatcttggc 960tcactgcaat
ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt
1020tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
tagagacggg 1080gtttcaccat attggccagg ctggtctcca actcctaatc
tcaggtgatc tacccacctt 1140ggcctcccaa attgctggga ttacaggcgt
gaaccactgc tcccttccct gtccttctga 1200ttttaaaata actataccag
caggaggacg tccagacaca gcataggcta cctggccatg 1260cccaaccggt
gggacatttg agttgcttgc ttggcactgt cctctcatgc gttgggtcca
1320ctcagtagat gcctgttgaa ttcgataccg tcgacctcga gggggggccc
ggtaccagct 1380tttgttccct ttagtgaggg ttaatttcga gcttggcgta
atcatggtca tagctgtttc 1440ctgtgtgaaa ttgttatccg ctcacaattc
cacacaacat acgagccgga agcataaagt 1500gtaaagcctg gggtgcctaa
tgagtgagct aactcacatt aattgcgttg cgctcactgc 1560ccgctttcca
gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
1620ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
tcgctgcgct 1680cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
ggcggtaata cggttatcca 1740cagaatcagg ggataacgca ggaaagaaca
tgtgagcaaa aggccagcaa aaggccagga 1800accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct ccgcccccct gacgagcatc 1860acaaaaatcg
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
1920cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
cttaccggat 1980acctgtccgc ctttctccct tcgggaagcg tggcgctttc
tcatagctca cgctgtaggt 2040atctcagttc ggtgtaggtc gttcgctcca
agctgggctg tgtgcacgaa ccccccgttc 2100agcccgaccg ctgcgcctta
tccggtaact atcgtcttga gtccaacccg gtaagacacg 2160acttatcgcc
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
2220gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
acagtatttg 2280gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
agttggtagc tcttgatccg 2340gcaaacaaac caccgctggt agcggtggtt
tttttgtttg caagcagcag attacgcgca 2400gaaaaaaagg atctcaagaa
gatcctttga tcttttctac ggggtctgac gctcagaaga 2460actcgtcaag
aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
2520gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca
cgggtagcca 2580acgctatgtc ctgatagcgg tccgccacac ccagccggcc
acagtcgatg aatccagaaa 2640agcggccatt ttccaccatg atattcggca
agcaggcatc gccatgggtc acgacgagat 2700cctcgccgtc gggcatgcgc
gccttgagcc tggcgaacag ttcggctggc gcgagcccct 2760gatgctcttc
gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
2820gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca
agcgtatgca 2880gccgccgcat tgcatcagcc atgatggata ctttctcggc
aggagcaagg tgagatgaca 2940ggagatcctg ccccggcact tcgcccaata
gcagccagtc ccttcccgct tcagtgacaa 3000cgtcgagcac agctgcgcaa
ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 3060cgtcctgcag
ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
3120cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt
tgtgcccagt 3180catagccgaa tagcctctcc acccaagcgg ccggagaacc
tgcgtgcaat ccatcttgtt 3240caatcatgcg aaacgatcct catcctgtct
cttgatcaga tcttgatccc ctgcgccatc 3300agatccttgg cggcaagaaa
gccatccagt ttactttgca gggcttccca accttaccag 3360agggcgcccc
agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagca
3420actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
gcgaaagggg 3480gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
ttcccagtca cgac 3534133534DNAartificial sequenceNucleic acid
sequence of the TV-GHRH plasmid. 13gttgtaaaac gacggccagt gaattgtaat
acgactcact atagggcgaa ttggagctcc 60accgcggtgg cggccgtccg ccctcggcac
catcctcacg acacccaaat atggcgacgg 120gtgaggaatg gtggggagtt
atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180tggcgctcta
aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca
240aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg
gccggggccg 300cattcctggg ggccgggcgg tgctcccgcc cgcctcgata
aaaggctccg gggccggcgg 360cggcccacga gctacccgga ggagcgggag
gcgccaagct ctagaactag tggatcccaa 420ggcccaactc cccgaaccac
tcagggtcct gtggacagct cacctagctg ccatggtgct 480ctgggtgttc
ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc
540ccctttgacc ctcaggatgc ggcggtatgt agatgccatc ttcaccaaca
gctaccggaa 600ggtgctggcc cagctgtccg cccgcaagct gctccaggac
atcctgaaca ggcagcaggg 660agagaggaac caagagcaag gagcataatg
actgcaggaa ttcgatatca agcttatcgg 720ggtggcatcc ctgtgacccc
tccccagtgc ctctcctggc cctggaagtt gccactccag 780tgcccaccag
ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct
840tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
gggaagacaa 900cctgtagggc ctgcggggtc tattgggaac caagctggag
tgcagtggca caatcttggc 960tcactgcaat ctccgcctcc tgggttcaag
cgattctcct gcctcagcct cccgagttgt 1020tgggattcca ggcatgcatg
accaggctca gctaattttt gtttttttgg tagagacggg 1080gtttcaccat
attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt
1140ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
gtccttctga 1200ttttaaaata actataccag caggaggacg tccagacaca
gcataggcta cctggccatg 1260cccaaccggt gggacatttg agttgcttgc
ttggcactgt cctctcatgc gttgggtcca 1320ctcagtagat gcctgttgaa
ttcgataccg tcgacctcga gggggggccc ggtaccagct 1380tttgttccct
ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc
1440ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
agcataaagt 1500gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
aattgcgttg cgctcactgc 1560ccgctttcca gtcgggaaac ctgtcgtgcc
agctgcatta atgaatcggc caacgcgcgg 1620ggagaggcgg tttgcgtatt
gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1680cggtcgttcg
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
1740cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
aaggccagga 1800accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
ccgcccccct gacgagcatc 1860acaaaaatcg acgctcaagt cagaggtggc
gaaacccgac aggactataa agataccagg 1920cgtttccccc tggaagctcc
ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1980acctgtccgc
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
2040atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
ccccccgttc 2100agcccgaccg ctgcgcctta tccggtaact atcgtcttga
gtccaacccg gtaagacacg 2160acttatcgcc actggcagca gccactggta
acaggattag cagagcgagg tatgtaggcg 2220gtgctacaga gttcttgaag
tggtggccta actacggcta cactagaaga acagtatttg 2280gtatctgcgc
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
2340gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
attacgcgca 2400gaaaaaaagg atctcaagaa gatcctttga tcttttctac
ggggtctgac gctcagaaga 2460actcgtcaag aaggcgatag aaggcgatgc
gctgcgaatc gggagcggcg ataccgtaaa 2520gcacgaggaa gcggtcagcc
cattcgccgc caagctcttc agcaatatca cgggtagcca 2580acgctatgtc
ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
2640agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
acgacgagat 2700cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag
ttcggctggc gcgagcccct 2760gatgctcttc gtccagatca tcctgatcga
caagaccggc ttccatccga gtacgtgctc 2820gctcgatgcg atgtttcgct
tggtggtcga atgggcaggt agccggatca agcgtatgca 2880gccgccgcat
tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
2940ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
tcagtgacaa 3000cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag
ccacgatagc cgcgctgcct 3060cgtcctgcag ttcattcagg gcaccggaca
ggtcggtctt gacaaaaaga accgggcgcc 3120cctgcgctga cagccggaac
acggcggcat cagagcagcc gattgtctgt tgtgcccagt 3180catagccgaa
tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
3240caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc
ctgcgccatc 3300agatccttgg cggcaagaaa gccatccagt ttactttgca
gggcttccca accttaccag 3360agggcgcccc agctggcaat tccggttcgc
ttgctgtcca taaaaccgcc cagtctagca 3420actgttggga agggcgatcg
gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 3480gatgtgctgc
aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac
3534143534DNAartificial sequenceNucleic acid sequence of the
15/27/28 GHRH plasmid. 14gttgtaaaac gacggccagt gaattgtaat
acgactcact atagggcgaa ttggagctcc 60accgcggtgg cggccgtccg ccctcggcac
catcctcacg acacccaaat atggcgacgg 120gtgaggaatg gtggggagtt
atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180tggcgctcta
aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca
240aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg
gccggggccg 300cattcctggg ggccgggcgg tgctcccgcc cgcctcgata
aaaggctccg gggccggcgg 360cggcccacga gctacccgga ggagcgggag
gcgccaagct ctagaactag tggatcccaa 420ggcccaactc cccgaaccac
tcagggtcct gtggacagct cacctagctg ccatggtgct 480ctgggtgttc
ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc
540ccctttgacc ctcaggatgc ggcggtatat cgatgccatc ttcaccaaca
gctaccggaa 600ggtgctggcc cagctgtccg cccgcaagct gctccaggac
atcctgaaca ggcagcaggg 660agagaggaac caagagcaag gagcataatg
actgcaggaa ttcgatatca agcttatcgg 720ggtggcatcc ctgtgacccc
tccccagtgc ctctcctggc cctggaagtt gccactccag 780tgcccaccag
ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct
840tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
gggaagacaa 900cctgtagggc ctgcggggtc tattgggaac
caagctggag tgcagtggca caatcttggc 960tcactgcaat ctccgcctcc
tgggttcaag cgattctcct gcctcagcct cccgagttgt 1020tgggattcca
ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg
1080gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
tacccacctt 1140ggcctcccaa attgctggga ttacaggcgt gaaccactgc
tcccttccct gtccttctga 1200ttttaaaata actataccag caggaggacg
tccagacaca gcataggcta cctggccatg 1260cccaaccggt gggacatttg
agttgcttgc ttggcactgt cctctcatgc gttgggtcca 1320ctcagtagat
gcctgttgaa ttcgataccg tcgacctcga gggggggccc ggtaccagct
1380tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca
tagctgtttc 1440ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
acgagccgga agcataaagt 1500gtaaagcctg gggtgcctaa tgagtgagct
aactcacatt aattgcgttg cgctcactgc 1560ccgctttcca gtcgggaaac
ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 1620ggagaggcgg
tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
1680cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
cggttatcca 1740cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
aggccagcaa aaggccagga 1800accgtaaaaa ggccgcgttg ctggcgtttt
tccataggct ccgcccccct gacgagcatc 1860acaaaaatcg acgctcaagt
cagaggtggc gaaacccgac aggactataa agataccagg 1920cgtttccccc
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
1980acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
cgctgtaggt 2040atctcagttc ggtgtaggtc gttcgctcca agctgggctg
tgtgcacgaa ccccccgttc 2100agcccgaccg ctgcgcctta tccggtaact
atcgtcttga gtccaacccg gtaagacacg 2160acttatcgcc actggcagca
gccactggta acaggattag cagagcgagg tatgtaggcg 2220gtgctacaga
gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
2280gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
tcttgatccg 2340gcaaacaaac caccgctggt agcggtggtt tttttgtttg
caagcagcag attacgcgca 2400gaaaaaaagg atctcaagaa gatcctttga
tcttttctac ggggtctgac gctcagaaga 2460actcgtcaag aaggcgatag
aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 2520gcacgaggaa
gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
2580acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg
aatccagaaa 2640agcggccatt ttccaccatg atattcggca agcaggcatc
gccatgggtc acgacgagat 2700cctcgccgtc gggcatgcgc gccttgagcc
tggcgaacag ttcggctggc gcgagcccct 2760gatgctcttc gtccagatca
tcctgatcga caagaccggc ttccatccga gtacgtgctc 2820gctcgatgcg
atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
2880gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg
tgagatgaca 2940ggagatcctg ccccggcact tcgcccaata gcagccagtc
ccttcccgct tcagtgacaa 3000cgtcgagcac agctgcgcaa ggaacgcccg
tcgtggccag ccacgatagc cgcgctgcct 3060cgtcctgcag ttcattcagg
gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 3120cctgcgctga
cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
3180catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat
ccatcttgtt 3240caatcatgcg aaacgatcct catcctgtct cttgatcaga
tcttgatccc ctgcgccatc 3300agatccttgg cggcaagaaa gccatccagt
ttactttgca gggcttccca accttaccag 3360agggcgcccc agctggcaat
tccggttcgc ttgctgtcca taaaaccgcc cagtctagca 3420actgttggga
agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
3480gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac
3534153534DNAartificial sequenceThis is the entire plasmid sequence
for wildtype GHRH. 15gttgtaaaac gacggccagt gaattgtaat acgactcact
atagggcgaa ttggagctcc 60accgcggtgg cggccgtccg ccctcggcac catcctcacg
acacccaaat atggcgacgg 120gtgaggaatg gtggggagtt atttttagag
cggtgaggaa ggtgggcagg cagcaggtgt 180tggcgctcta aaaataactc
ccgggagtta tttttagagc ggaggaatgg tggacaccca 240aatatggcga
cggttcctca cccgtcgcca tatttgggtg tccgccctcg gccggggccg
300cattcctggg ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg
gggccggcgg 360cggcccacga gctacccgga ggagcgggag gcgccaagct
ctagaactag tggatcccaa 420ggcccaactc cccgaaccac tcagggtcct
gtggacagct cacctagctg ccatggtgct 480ctgggtgttc ttctttgtga
tcctcaccct cagcaacagc tcccactgct ccccacctcc 540ccctttgacc
ctcaggatgc ggcggtatgc agatgccatc ttcaccaaca gctaccggaa
600ggtgctgggc cagctgtccg cccgcaagct gctccaggac atcatgagca
ggcagcaggg 660agagaggaac caagagcaag gagcataatg actgcaggaa
ttcgatatca agcttatcgg 720ggtggcatcc ctgtgacccc tccccagtgc
ctctcctggc cctggaagtt gccactccag 780tgcccaccag ccttgtccta
ataaaattaa gttgcatcat tttgtctgac taggtgtcct 840tctataatat
tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa
900cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
caatcttggc 960tcactgcaat ctccgcctcc tgggttcaag cgattctcct
gcctcagcct cccgagttgt 1020tgggattcca ggcatgcatg accaggctca
gctaattttt gtttttttgg tagagacggg 1080gtttcaccat attggccagg
ctggtctcca actcctaatc tcaggtgatc tacccacctt 1140ggcctcccaa
attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga
1200ttttaaaata actataccag caggaggacg tccagacaca gcataggcta
cctggccatg 1260cccaaccggt gggacatttg agttgcttgc ttggcactgt
cctctcatgc gttgggtcca 1320ctcagtagat gcctgttgaa ttcgataccg
tcgacctcga gggggggccc ggtaccagct 1380tttgttccct ttagtgaggg
ttaatttcga gcttggcgta atcatggtca tagctgtttc 1440ctgtgtgaaa
ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
1500gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
cgctcactgc 1560ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
atgaatcggc caacgcgcgg 1620ggagaggcgg tttgcgtatt gggcgctctt
ccgcttcctc gctcactgac tcgctgcgct 1680cggtcgttcg gctgcggcga
gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1740cagaatcagg
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
1800accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
gacgagcatc 1860acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
aggactataa agataccagg 1920cgtttccccc tggaagctcc ctcgtgcgct
ctcctgttcc gaccctgccg cttaccggat 1980acctgtccgc ctttctccct
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2040atctcagttc
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
2100agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
gtaagacacg 2160acttatcgcc actggcagca gccactggta acaggattag
cagagcgagg tatgtaggcg 2220gtgctacaga gttcttgaag tggtggccta
actacggcta cactagaaga acagtatttg 2280gtatctgcgc tctgctgaag
ccagttacct tcggaaaaag agttggtagc tcttgatccg 2340gcaaacaaac
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
2400gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
gctcagaaga 2460actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc
gggagcggcg ataccgtaaa 2520gcacgaggaa gcggtcagcc cattcgccgc
caagctcttc agcaatatca cgggtagcca 2580acgctatgtc ctgatagcgg
tccgccacac ccagccggcc acagtcgatg aatccagaaa 2640agcggccatt
ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
2700cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc
gcgagcccct 2760gatgctcttc gtccagatca tcctgatcga caagaccggc
ttccatccga gtacgtgctc 2820gctcgatgcg atgtttcgct tggtggtcga
atgggcaggt agccggatca agcgtatgca 2880gccgccgcat tgcatcagcc
atgatggata ctttctcggc aggagcaagg tgagatgaca 2940ggagatcctg
ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
3000cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
cgcgctgcct 3060cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt
gacaaaaaga accgggcgcc 3120cctgcgctga cagccggaac acggcggcat
cagagcagcc gattgtctgt tgtgcccagt 3180catagccgaa tagcctctcc
acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 3240caatcatgcg
aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc
3300agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca
accttaccag 3360agggcgcccc agctggcaat tccggttcgc ttgctgtcca
taaaaccgcc cagtctagca 3420actgttggga agggcgatcg gtgcgggcct
cttcgctatt acgccagctg gcgaaagggg 3480gatgtgctgc aaggcgatta
agttgggtaa cgccagggtt ttcccagtca cgac 3534
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