U.S. patent application number 10/262377 was filed with the patent office on 2003-08-07 for super-active porcine growth hormone releasing hormone analog.
This patent application is currently assigned to Baylor College of Medicine. Invention is credited to Draghia-Alki, Ruxandra, Schwartz, Robert J..
Application Number | 20030148948 10/262377 |
Document ID | / |
Family ID | 22513900 |
Filed Date | 2003-08-07 |
United States Patent
Application |
20030148948 |
Kind Code |
A1 |
Schwartz, Robert J. ; et
al. |
August 7, 2003 |
Super-active porcine growth hormone releasing hormone analog
Abstract
Inadequate growth due to deficiencies in growth hormone (GR),
growth hormone releasing hormone (GHRH), or genetic diseases can be
ameliorated utilizing recombinant protein therapy with a novel GHRH
analog having a sequence (SEQ ID NO:1). Also included is (1) a
method of treating growth hormone-related deficiencies associated
with the growth hormone pathway; (2) a method for treating growth
hormone-related deficiencies associated with genetic disease; (3) a
method to improve growth performance in an animal; (4) a method of
treating an animal having a growth deficiency disease; (5) a method
of increasing the efficiency of an animal used for food; and. (6) a
method to enhance growth in an animal.
Inventors: |
Schwartz, Robert J.;
(Houston, TX) ; Draghia-Alki, Ruxandra; (Houston,
TX) |
Correspondence
Address: |
JACKSON WALKER LLP
2435 NORTH CENTRAL EXPRESSWAY
SUITE 600
RICHARDSON
TX
75080
US
|
Assignee: |
Baylor College of Medicine
|
Family ID: |
22513900 |
Appl. No.: |
10/262377 |
Filed: |
October 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10262377 |
Oct 1, 2002 |
|
|
|
09624268 |
Jul 24, 2000 |
|
|
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60145624 |
Jul 26, 1999 |
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Current U.S.
Class: |
514/11.2 ;
435/320.1; 435/455; 514/11.4; 514/16.5; 514/3.8; 514/44R;
514/8.6 |
Current CPC
Class: |
A61P 5/06 20180101; A61P
17/02 20180101; A61K 38/00 20130101; C07K 14/60 20130101; A61P 5/00
20180101; A61P 31/18 20180101; A61K 48/00 20130101; A61P 3/00
20180101 |
Class at
Publication: |
514/12 ; 514/44;
435/320.1; 435/455 |
International
Class: |
A61K 048/00; A61K
038/25; C12N 015/85 |
Claims
What is claimed:
1. As a composition of matter, growth hormone-releasing hormone
having an amino acid sequence of SEQ ID NO: 1.
2. A pharmaceutical composition for stimulating the release of
growth hormone in animals comprising the growth hormone releasing
hormone of claim 1 in a pharmaceutically acceptable carrier.
3. As a composition of matter, a nucleotide sequence encoding the
growth hormone-releasing hormone having the amino acid sequence of
SEQ ID NO:1.
4. A vector comprising a promoter; a nucleotide sequence encoding
SEQ ID NO:1; and a 3' untranslated region operatively linked for
functional expression.
5. The vector of claim 4, wherein said promoter is a synthetic
myogenic promoter.
6. The vector of claim 4, wherein said 3' untranslated region is
the hGH 3' untranslated region.
7. The method of increasing growth hormone in an animal comprising
the step of introducing into said animal a therapeutically
effective amount of the vector of claim 4.
8. The method of treating in an animal a growth hormone-related
deficiency disease associated with the growth hormone pathway
comprising the step of introducing into said animal a
therapeutically effective amount of the vector of claim 4.
9. The method of claim 8, wherein said deficiency disease is the
result of a change in the genetic material in said animal.
10. The method of improving growth performance in an animal
comprising the step of introducing a therapeutically effective
amount of the vector of claim 4.
11. The method of increasing the efficiency of an animal comprising
the step of introducing a therapeutically effective amount of the
vector of claim 4.
12. The method of treating wasting symptoms in an animal, wherein
said wasting symptoms are associated with burn, trauma, AIDS or
other consumption diseases, comprising the step of introducing a
therapeutically effective amount of the vector of claim 4.
13. The method of enhancing growth in an animal comprising the step
of introducing a therapeutically effective amount of the vector of
claim 4.
14. The method of treating a growth hormone-related deficiency
disease associated with the growth hormone pathway comprising the
step of introducing a therapeutically effective amount of the
vector of claim 4.
15. The method of claim 7, 8, 9, 10, 11, 12, 13 or 14, wherein the
animal is selected from the group consisting of a human, a pet
animal, a food animal and a work animal.
16. The method of claim 7, 8, 9, 10, 11, 12, 13 or 14, wherein said
vector is introduced into myogenic cells.
17. The method of claim 7, 8, 9, 10, 11, 12, 13 or 14, wherein said
vector is introduced into muscle tissue of said animal.
18. The method of claim 7, 8, 9, 10, 11, 12, 13 or 14, wherein said
vector is introduced into said animal in a single
administration.
19. The vector of claim 4, wherein said vector is selected from the
group consisting of a plasmid, a viral vector, a liposome, and a
cationic lipid.
20. A method of treating growth hormone-related deficiencies
associated with the growth hormone pathway in an animal comprising
the step of introducing a therapeutically effective amount of a
vector into an animal, said vector comprised of a synthetic
myogenic promoter; a nucleotide sequence encoding SEQ ID NO:1; and
the 3' untranslated region of hGH operatively linked for functional
expression.
21. A method for stimulating production of growth hormone in an
animal at a level greater than that associated with normal growth,
said method comprising introducing into said animal a
therapeutically effective amount of a vector, said vector
comprising a synthetic myogenic promoter; a nucleotide sequence
encoding SEQ ID NO:1; and a 3' untranslated region of hGH
operatively linked for functional expression.
Description
[0001] This application claims priority to U.S. Provisional
Application Serial No. 60/145,624 filed Jul. 26, 1999.
FIELD OF THE INVENTION
[0002] This invention relates to the treatment of growth
deficiencies; the improvement of growth performance; the
stimulation of production of growth hormone in an animal at a level
greater than that associated with normal growth; and the
enhancement of growth utilizing the administration of a growth
hormone releasing hormone analog. Furthermore it relates to the
application of a nucleotide sequence encoding said growth hormone
releasing hormone analog regulated by a muscle-specific promoter
into muscle tissue, particularly using gene therapy techniques.
BACKGROUND OF THE INVENTION
[0003] The growth hormone (GH) 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) agonists and
antagonists, such as growth hormone releasing hormone (GHRH) and
somatostatin, 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 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, both
hypothalamic hormones. The central role of GH in controlling
somatic growth in humans and other vertebrates, and the
physiologically relevant pathways regulating GH secretion from the
pituitary are well known. GH increases production of IGF-I,
primarily in the liver, and other target organs. IGF-I and GH, in
turn, feedback on the hypothalamus and pituitary to inhibit GHRH
and GH release. GH has both direct and indirect actions on
peripheral tissues, the indirect effects being mediated mainly by
IGF-I.
[0004] There is a wide spectrum of clinical conditions, both in
children and adults, in which linear growth (prepubertal patients)
or body composition are compromised, and which respond to GH or
GHRH therapy. In all instances the GHRH-GH-IGF-I axis is
functional, but not necessarily operating at optimal sensitivity or
responsiveness for a variety of possible reasons.
[0005] The principal feature of GH deficiencies in children is
short stature. Similar phenotypes are produced by genetic defects
at different points in the GH axis (Parks et al., 1995), as well as
non-GH-deficient short stature. Non-GH-deficiencies have different
etiology: (1) genetic diseases, Turner syndrome (Jacobs et al.,
1990; Skuse et al., 1999), hypochondroplasia (Tanaka et al., 1998;
Key and Gross, 1996), and Crohn's disease (Savage et al., 1999);
and (2) intrauterine growth retardation (Albanese and Stanhope,
1997; Azcona et al., 1998); and (3) chronic renal insufficiency
(Sohmiya et al., 1998; Benfield and Kohaut, 1997). Cases where the
GH axis is unaffected (i.e. patients have normal hormones, genes
and receptors) account for more than 50% of the total cases of
growth retardation. In these cases GHRH or GH therapy has been
shown to be effective (Gesundheit and Alexander, 1995).
[0006] Reduced GH secretion from the anterior pituitary causes
skeletal muscle mass to be lost during aging from 25 years to
senescence. The GHRH-GH-IGF-1 axis undergoes dramatic changes
through aging and in the elderly (D'Costa et al., 1993) with
decreased GH production rate and GH half-life, decreased IGF-1
response to GH and GHRH stimuli leading to loss of skeletal muscle
mass (sarcopenia), osteoporosis, and increase in fat and decrease
in lean body mass (Bartke, 1998). Previous studies have shown that
in a significant number of normal elderly persons, GH and IGFs
levels in serum are significantly reduced by 70-80% of their
teenage level (Corpas et al., 1993; Iranmanesh et al., 1991). It
has been demonstrated that the development of sarcopenia can be
offset by GH therapy. However, this remains a controversial therapy
in the elderly because of its cost and frequent side effects.
[0007] The production of recombinant proteins allows a useful tool
for the treatment of these conditions. Although GH replacement
therapy is widely used in patients with growth deficiencies and
provides satisfactory growth, and may have positive psychological
effects on the children being treated (Rosenbaum and Saigal, 1996;
Erling, 1999), this therapy has several disadvantages, including an
impractical requirement for frequent administration of GH (Monti et
al., 1997; Heptulla et al., 1997) and undesirable secondary effects
(Blethen et al., 1996; Watkins, 1996; Shalet et al., 1997; Allen et
al, 1997).
[0008] It is well established that 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-1 levels,
increases GH secretion proportionally to the GHRH dose, yet still
invokes a response to bolus doses of GHRH (Bercu and Walker, 1997).
Thus, GHRH administration represents a more physiological
alternative of increasing subnormal GH and IGF-1 levels (Corpas et
al., 1993).
[0009] Although 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, GHRH
administration is not practical. However, extracranially secreted
GHRH, as a processed protein species (Tyr1-40 or Tyr1-Leu44) or
even as shorter truncated molecules, are biologically active
(Thorner et al., 1984). Importantly, a low level of GHRH (100
pg/ml) in the blood supply stimulates GH secretion (Corpas et al.,
1993) and makes GHRH an excellent candidate for gene therapeutic
expression. 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 a preferred 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 modest
extent over a period of two weeks (Draghia-Akli et al., 1997).
[0010] 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)NH.sub.2 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). Incorporating cDNA coding for the shorter
GHRH, species (1-40)0H, in a gene therapy vector might result in a
molecule with a longer half-life in serum, increased potency, and
will provide greater GH release in plasmid injected animals. In
addition, mutagenesis via amino acid replacement of protease
sensitive amino acids could prolong the serum half-life of the
hGHRH molecule. Furthermore, the enhancement of biological activity
of GHRH is achieved by using super-active analogs which may
increase its binding affinity to specific receptors.
[0011] There are issued patents which address 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. A GHRH analog containing the following mutations
has been reported (U.S. Pat. No. 5,846,936): Tyr at position 1 to
His; Ala at position 2 to Val, Leu, or others; Asn at position 8 to
Gln, Ser, 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 analog of the
present invention does not contain all of the amino acid
substitutions reported in U.S. Pat. No. 5,846,936 to be necessary
for activity.
[0012] Although specific embodiments of U.S. Pat. No. 5,756,264
concern gene therapy wherein the therapeutic gene is delivered into
myogenic tissue, and one example mentioned in the specification is
growth hormone releasing hormone, two important differences
differentiate this system from the present invention. First, this
invention concerns an analog of growth hormone releasing hormone
which differs from the wild type form with significant
modifications which improve its function as a GH secretagogue:
decreased susceptibility to proteases and increased stability,
which would prolong the ability to effect a therapy, and increased
biological activity, which would enhance the ability to effect a
therapy. In addition, in one aspect of the present invention it
utilizes a unique synthetic promoter, termed SPc5-12 (Li et al.,
1999), which contains a proximal serum response element (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. The uniqueness of such a
synthetic promoter is a significant improvement over, for instance,
issued patents concerning a myogenic promoter and its use (e.g.
U.S. Pat. No. 5,374,544) or systems for myogenic expression of a
nucleic acid sequence (e.g. U.S. Pat. No. 5,298,422).
[0013] Thus, the present invention teaches application of an analog
containing mutations which improve the ability to elicit the
release of growth hormone. As illustrated in the Examples, said
analog succeeds in increasing release of growth hormone despite the
absence of the substitution at position 8 to Gln, Ser, or Thr in
the analog of the prior art. Furthermore, it provides gene therapy
techniques to introduce said analog, whose expression is regulated
by a synthetic myogenic promoter, into the preferred choice of
skeletal muscle tissue since muscle fiber has a long life span and
can be transduced by circular DNA plasmids. This is an improvement
over the present art, in which the requirement for frequent
administration of GHRH protein precludes it for use as a chronic
treatment.
SUMMARY OF THE INVENTION
[0014] An embodiment of the present invention is the growth hormone
releasing hormone having the amino acid sequence of SEQ ID NO:
1.
[0015] Additional embodiments of the present invention include: (1)
a method for treating growth hormone-related deficiencies
associated with the growth hormone pathway; (2) a method for
treating growth hormone-related deficiencies associated with
genetic disease; (3) a method to improve growth performance in an
animal; (4) a method of treating an animal having a growth
deficiency disease; (5) a method of increasing the efficiency of an
animal used for food; and (6) a method of treating in an animal
wasting symptoms associated with burn, trauma, AIDS, or other
consumption diseases; (7) a method for stimulating production of
growth hormone in an animal at a level greater than that associated
with normal growth; and (8) a method of enhancing growth in an
animal. All of these methods include the step of introducing a
plasmid vector into an animal, wherein said vector comprises a
promoter; a nucleotide sequence encoding SEQ ID NO:1; and a 3'
untranslated region operatively linked sequentially at appropriate
distances for functional expression.
[0016] In a preferred embodiment the promoter is a synthetic
myogenic promoter and hGH 3' untranslated region is in the 3'
untranslated region.
[0017] In specific embodiments said vector 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.
[0018] An additional embodiment is a pharmaceutical composition for
stimulating the release of growth hormone in animals comprising SEQ
ID NO:1 in a pharmaceutically acceptable carrier.
[0019] Another embodiment of the present invention is the
nucleotide sequence encoding the growth hormone-releasing hormone
having the amino acid sequence of SEQ ID NO:1.
[0020] In an additional embodiment of the present invention there
is a method of increasing growth hormone in an animal comprising
the step of introducing a therapeutically effective amount of a
vector into an animal, said vector comprised of a promoter; a
nucleotide sequence encoding SEQ ID NO:1; and a 3' untranslated
region operatively linked for functional expression. In a specific
embodiment the promoter is a synthetic myogenic promoter. In
another specific embodiment the 3' untranslated region is the hGH
3' untranslated region. In another specific embodiment the animal
is selected from the group consisting of a human, a pet animal, a
food animal and a work animal. In an additional specific embodiment
the vector is introduced into myogenic cells. In a further specific
embodiment the vector is introduced into muscle tissue of said
animal. In another specific embodiment the introduction treats a
growth hormone-related deficiency disease associated with the
growth hormone pathway. In an additional specific embodiment the
deficiency disease is the result of a change in the genetic
material in said animal. In a further embodiment the introduction
results in improving growth performance in said animal. In another
embodiment the introduction increases the efficiency of the animal,
wherein the animal is used for food. In an additional embodiment
the introduction treats in an animal wasting symptoms associated
with burn, trauma, AIDS, or other consumption diseases. In another
specific embodiment the introduction results in enhancement of
growth of said animal. In another specific embodiment the vector is
introduced into said animal in a single administration. In a
further specific embodiment the vector is selected from the group
consisting of a plasmid, a viral vector, a liposome, and a cationic
lipid.
[0021] In an additional embodiment of the present invention there
is a method of treating growth hormone-related deficiencies
associated with the growth hormone pathway in an animal comprising
the step of introducing a therapeutically effective amount of a
vector into an animal, said vector comprised of a synthetic
myogenic promoter; a nucleotide sequence encoding SEQ ID NO: 1; and
the 3' untranslated region of hGH operatively linked for functional
expression.
[0022] In another embodiment of the present invention there is a
method for stimulating production of growth hormone in an animal at
a level greater than that associated with normal growth, said
method comprising introducing into said animal an effective amount
of a vector, said vector comprising a synthetic myogenic promoter;
a nucleotide sequence encoding SEQ ID NO:1; and a 3' untranslated
region of hGH operatively linked for functional expression.
[0023] Other and further objects, features and advantages would be
apparent and eventually more readily understood by reading the
following specification and by reference to the company drawing
forming a part thereof, or any examples of the presently preferred
embodiments of the invention are given for the purpose of the
disclosure.
DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A through FIG. 1C demonstrate that GHRH super-active
analogs increase GH secretagogue activity and stability. FIG. 1A is
a comparison of the porcine wild type (1-40)OH amino acid sequence
with the analog HV-GHRH. FIG. 1B shows the effect of the different
GHRH species on pig GH release in porcine primary pituitary
culture. FIG. 1C demonstrates changes in stability which occur with
HV-GHRH and wild type porcine GHRH during a 4 to 6 hour
incubation.
[0025] FIG. 2A through FIG. 2E demonstrate an increase in GHRH, GH
and IGF-I serum levels over two months following single injections
of super-active analog GHRH myogenic expression vector. FIG. 2A
depicts the constructs which contain the SPc5-12 synthetic promoter
and the 3' UTR of GH. As a model of mutated protein, HV-GHRH
construct was used and compared with the porcine wild type as a
positive control, and with .beta.-galactosidase construct as a
negative control. FIG. 2B illustrates relative levels of serum GHRH
in pSP-GHRH injected pigs versus placebo injected control pigs.
FIG. 2C demonstrates absolute levels of serum GHRH in pSP-GHRH
injected pigs versus controls pigs corrected for weight/blood
volume increase. FIG. 2D shows variation of GH levels in
pSP-HV-GHRH injected pigs. FIG. 2E shows plasma IGF-1 levels
following direct intramuscular injection of pSP-GHRH
constructs.
[0026] FIG. 3A through FIG. 3C demonstrate the effect of myogenic
GHRH expression vectors on pig growth. FIG. 3A shows the change in
average weight in injected pigs over 2 months with pSP-GHRH or
pSP-GHRH-HV. FIG. 3B shows the status of feed efficiency in the
pSP-GHRH injected pigs versus controls. FIG. 3C is a comparison of
a pSP-HV-GHRH injected pig and a placebo injected control pig, 45
days post-injection.
[0027] FIG. 4 demonstrates the effect of injection of different
amounts of pSP-GHRH-HV on 10 day-old piglets.
[0028] FIG. 5 shows the effect of injection of different amounts of
pSP-GHRH-HV on IGF-I levels in 10 day-old piglets.
[0029] FIG. 6 illustrates a time course for pSP-GHRH-HV plasmid
injection into piglets.
DETAILED DESCRIPTION OF THE INVENTION
[0030] 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.
[0031] The term "animal" as used herein refers to any species of
the animal kingdom. In preferred embodiments it refers more
specifically to humans, animals used as pets (dogs, cats, horses),
animals used for work (horses, cows) and food animals which include
animals which produce food (chickens, cows, fish) or are themselves
food (frogs, chickens, fish, crabs, lobsters, shrimp, mussels,
scallops, goats, boars, cows, lambs, pigs, ostrich, emu, eel) and
others well known in the art.
[0032] The term "consumption diseases" as used herein are defined
as diseases in which one loses weight (mostly muscle mass), loses
muscle strength, may have demineralization of bones (involuntary,
with no known mechanism), may have a combination of viral/bacterial
infection, or may have deregulation of some basic metabolisms. Some
examples of such diseases are AIDS, tuberculosis, or cancer.
[0033] 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 end-points known to those
skilled in the art.
[0034] The term "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.
[0035] 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.
[0036] 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.
[0037] The term "growth hormone releasing hormone" as used herein
is defined as a hormone which facilitates or stimulates release of
growth hormone.
[0038] The term "growth hormone releasing hormone analog" as used
herein is defined as a protein which contains amino acid mutations
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.
[0039] The term "myogenic" as used herein refers specifically to
muscle tissue.
[0040] The term "pharmaceutically acceptable" as used herein refers
to a compound wherein administration of said compound can be
tolerated by a recipient mammal.
[0041] The term "secretagogue" as used herein refers to a natural
or synthetic molecule that enhances synthesis and secretion of a
downstream--regulated molecule (e.g. GHRH is a secretagogue for
GH).
[0042] 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. A skilled artisan is aware that a
sufficient vector amount is utilized to provide expression of a
nucleotide sequence encoding SEQ ID NO:1 to therapeutically
effective levels.
[0043] The term "treats" as used herein is defined as the act of
affecting favorably at least one symptom of a growth deficiency
disease or affecting favorably the growth of an animal. A skilled
artisan is aware that the term "treats" does not necessarily
indicate cure, although a cure of the symptom or symptoms is within
the scope of the term treat.
[0044] 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.
[0045] The term "wasting symptoms" as used herein is defined as a
condition associated with consumption diseases.
[0046] An embodiment of the present invention is the growth
hormone-releasing hormone analog having the amino acid sequence of
SEQ ID NO:1 and all nucleotide sequences encoding same.
[0047] Additional embodiments of the present invention include: (1)
a method for treating growth hormone-related deficiencies
associated with the growth hormone pathway; (2) a method for
treating growth hormone-related deficiencies associated with
genetic disease; (3) a method to improve growth performance in an
animal; (4) a method of treating an animal having a growth
deficiency disease; (5) a method of increasing the efficiency of an
animal used for food; (6) a method of treating in an animal wasting
symptoms associated with burn, trauma, AIDS, or other consumption
diseases; (7) a method for stimulating production of growth hormone
in an animal at a level greater than that associated with normal
growth; and (8) a method of enhancing growth in an animal. All of
these methods include the step of introducing a plasmid vector into
an animal, wherein said vector comprises a promoter; a nucleotide
sequence encoding SEQ ID NO:1; and a 3' untranslated region
operatively linked sequentially at appropriate distances for
functional expression. In a specific embodiment these methods
result in increasing, improving or enhancing growth, or they result
in an increase of the production of growth hormone.
[0048] In a specific embodiment there is a method of treating
growth hormone-related deficiencies associated with the growth
hormone pathway in an animal comprising the step of introducing a
therapeutically effective amount of a vector into an animal, said
vector comprised of a promoter; a nucleotide sequence encoding SEQ
ID NO:1; and a 3' untranslated region operatively linked
sequentially at appropriate distances for functional expression. A
skilled artisan is aware that such deficiencies in the growth
hormone pathway may affect it indirectly or directly, and the step
affected may be upstream or downstream of GHRH action or function.
In a specific embodiment in which a downstream step from GHRH
action or function is affected, elevated levels of the GHRH analog
of the present invention, originally administered in gene therapy
form, overcomes this affected step.
[0049] In another specific embodiment there is a method of treating
growth hormone-related deficiencies associated with genetic disease
in an animal comprising the step of introducing a therapeutically
effective amount of a vector into an animal, said vector comprised
of a promoter; a nucleotide sequence encoding SEQ ID NO:1; and a 3'
untranslated region operatively linked sequentially at appropriate
distances for functional expression. The deficiency may be directly
or indirectly caused by the genetic disease, and other phenotypes
may also be present. Examples of genetic diseases include but are
not limited to Creutzfeldt-Jakob disease, Cohen syndrome,
aminopterin-methotrexate syndrome, Kabuki syndrome, Wolf-Hirschhorn
syndrome, Russell-Silver syndrome, Miller-Dieker syndromes,
Langerhans cell histiocytosis, Roberts syndrome, and
18q-syndrome.
[0050] In another embodiment there is a method of treating in an
animal having a growth deficiency disease comprising the step of
introducing a therapeutically effective amount of a vector into an
animal, said vector comprised of a promoter; a nucleotide sequence
encoding SEQ ID NO:1; and a 3' untranslated region operatively
linked sequentially at appropriate distances for functional
expression. The growth deficiency disease may be due to a genetic
defect or due to a deficiency in the growth hormone pathway.
[0051] In another embodiment of the present invention there is a
method of improving growth performance in an animal comprising the
step of introducing an effective amount of a vector into cells of
said animal, said vector comprised of a promoter; a nucleotide
sequence encoding SEQ ID NO:1; and a 3' untranslated region
operatively linked sequentially at appropriate distances for
functional expression. The term "growth performance" as used herein
is defined as the state or status of growth of an animal. The
growth performance may be as a result of a genetic disease, a
growth related deficiency, or exposure to a growth-affecting agent,
either of the animal or of a parent of the animal. The method of
improving growth performance in an animal in a specific embodiment
comprises the method of increasing growth of the animal.
[0052] In an additional specific embodiment there is a method for
stimulating production of growth hormone in an animal at a level
greater than that associated with normal growth, said method
comprising introducing into said animal an effective amount of a
vector, said vector comprising a promoter; a nucleotide sequence
encoding SEQ ID NO:1; and a 3' untranslated region operatively
linked sequentially at appropriate distances for functional
expression. A level greater than that associated with normal growth
includes the basal, inherent growth of an animal with a
growth-related deficiency or of an animal with growth levels
similar to other similar animals in the population, including those
with no growth-related deficiency.
[0053] In another embodiment there is a method of enhancing growth
in an animal comprising introducing into said animal an effective
amount of a vector, said vector comprising a promoter; a nucleotide
sequence encoding SEQ ID NO:1; and a 3' untranslated region
operatively linked sequentially at appropriate distances for
functional expression. The animal whose growth is enhanced may or
may not have a growth deficiency.
[0054] In an embodiment of the present invention there is a vector
comprised of a promoter; a nucleotide sequence encoding SEQ ID
NO:1; and a 3' untranslated region operatively linked sequentially
at appropriate distances for functional expression. One skilled in
the art recognizes that a variety of nucleotide sequences can be
used to encode SEQ ID NO:1. The specific sequence to be used is
partially determined on specific sequences to be modified and the
experimental conditions determined by the skilled artisan for the
specific use. As shown herein the skilled artisan can use a GHRH
cDNA sequence for site-directed mutagenesis to create changes in
the sequence to contain both the native or species-specific
sequence and the desired amino acid substitutions for protease
resistance, etc. Examples provided herein are directed toward how
to alter the nucleotide sequence by methods such as site-directed
mutagenesis to obtain the desired sequence. A skilled artisan is
thus aware how to obtain a nucleotide sequence encoding SEQ ID NO:1
by utilizing, for example, SEQ ID NO:8 or a similar sequence from
GenBank (see below) as a template to make alterations to it by
site-directed mutagenesis or other known methods to obtain
nucleotide sequence which encodes SEQ ID NO:1. The amino acid
sequence of SEQ ID NO:1, which is encoded by multiple nucleotide
sequences due to the wobble (third) position of each codon, could
be easily created by a skilled artisan given the access to GenBank
for sequence, the methods provided herein for site-directed
mutagenesis, and a codon table for the genetic code, such as is
found in any standard biochemistry or molecular biology textbook
(e.g. Biochemistry, 3.sup.rd ed., L. Stryer; W. H. Freeman and Co.,
N.Y. (1988)).
[0055] In a preferred embodiment the promoter is a synthetic
myogenic promoter and hGH 3' untranslated region is in the 3'
untranslated region. In a specific embodiment of the present
invention there is utilized a synthetic promoter, termed SPc5-12
(Li et al., 1999) (SEQ ID NO:6), which contains a proximal serum
response element (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. 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
(http://www.ncbi.nlm.nih.gov/Genbank/- GenbankSearch.html) or the
NCBI PubMed site(http://www.ncbi.nlm.nih.gov/Pu- bMed/). 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.
[0056] In a specific embodiment the hGH 3' untranslated region (SEQ
ID NO:7) is utilized in a nucleic acid vector, such as a
plasmid.
[0057] In a specific embodiment there is a method to increase
growth hormone in an animal utilizing a vector comprising
nucleotide sequence encoding SEQ ID NO:1. As described in the
Examples, human GHRH cDNA (SEQ ID NO:8) is used as a template for
site-directed mutagenesis to create changes of the sequence to
contain both the native porcine sequence and the desired amino acid
substitutions for protease resistance, etc. Thus, the Examples
provide teachings herein regarding how to alter the nucleotide
sequence by methods such as site-directed mutagenesis to obtain the
desired sequence. A skilled artisan is thus aware how to obtain a
nucleotide sequence encoding SEQ ID NO:1 by utilizing, for example,
SEQ ID NO:8 or a similar sequence from GenBank (see supra) as a
template to make alterations to it by site-directed mutagenesis or
other known methods to obtain nucleotide sequence which encodes SEQ
ID NO:1. The amino acid sequence of SEQ ID NO:1, which is encoded
by multiple nucleotide sequences due to the wobble (third) position
of each codon, could be easily created by a skilled artisan given
the access to GenBank for sequence, the methods provided herein for
site-directed mutagenesis, and a codon table for the genetic code,
such as is found in any standard biochemistry or molecular biology
textbook (e.g. Biochemistry, 3.sup.rd ed., L. Stryer; W. H. Freeman
and Co., N.Y. (1988)).
[0058] In specific embodiments said vector 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.
[0059] An additional embodiment is a pharmaceutical composition for
stimulating the release of growth hormone in animals comprising SEQ
ID NO:1 in a pharmaceutically acceptable carrier.
[0060] Another embodiment of the present invention is the
nucleotide sequence encoding the growth hormone-releasing hormone
having the amino acid sequence of SEQ ID NO:1.
[0061] In addition to the specific embodiment of introducing said
construct into the animal via a plasmid vector, delivery systems
for tranfection 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, 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.
[0062] 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 which 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.
[0063] Nucleic Acids
[0064] 1. Vectors
[0065] 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 it can be replicated. 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.
[0066] The term "expression vector" refers to a vector 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.
[0067] a. Promoters and Enhancers
[0068] 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.
[0069] A promoter may be one naturally associated with a gene or
sequence, as may be obtained by isolating the 5' non-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., in connection
with the compositions disclosed herein (see U.S. Pat. No.
4,683,202, U.S. Pat. No. 5,928,906, each incorporated herein by
reference). 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.
[0070] 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, for example, see Sambrook et al. (1989), incorporated
herein by reference. 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).
[0071] 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 (Nomoto et al. 1999), the somatostatin receptor 2
gene (Kraus et al., 1998), murine epididymal retinoic acid-binding
gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998),
mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), D1A dopamine
receptor gene (Lee, et al., 1997), insulin-like growth factor II
(Wu et al., 1997), human platelet endothelial cell adhesion
molecule-1 (Almendro et al., 1996).
[0072] b. Initiation Signals and Internal Ribosome Binding
Sites
[0073] 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.
[0074] 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 (Pelletier and Sonenberg,
1988). IRES elements from two members of the picornavirus family
(polio and encephalomyocarditis) have been described (Pelletier and
Sonenberg, 1988), as well an IRES from a mammalian message (Macejak
and Sarnow, 1991). 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 (see U.S. Pat. Nos. 5,925,565 and
5,935,819, herein incorporated by reference).
[0075] c. Multiple Cloning Sites
[0076] 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. (See Carbonelli et
al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated
herein by reference.) "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.
[0077] d. Splicing Sites
[0078] 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. (See Chandler et al., 1997,
herein incorporated by reference.)
[0079] e. Polyadenylation Signals
[0080] 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 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.
[0081] f. Origins of Replication
[0082] 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.
[0083] g. Selectable and Screenable Markers
[0084] 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.
[0085] 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.
[0086] 2. Host Cells
[0087] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these term 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
organisms 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.
[0088] 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 Gold Cells (STRATAGENE.RTM., La Jolla). Alternatively,
bacterial cells such as E. coli LE392 could be used as host cells
for phage viruses.
[0089] Examples of eukaryotic host cells for replication and/or
expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO,
Saos, and PC12. 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.
[0090] 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.
[0091] 3. Expression Systems
[0092] 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.
[0093] The insect cell/baculovirus system can produce a high level
of protein expression of a heterologous nucleic acid segment, such
as described in U.S. Pat. Nos. 5,871,986, 4,879,236, both herein
incorporated by reference, 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..
[0094] Other examples of expression systems include
STRATAGENE.RTM.'s COMPLETE CONTROL 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.
[0095] Mutagenesis
[0096] 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.
[0097] 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.
[0098] Site-Directed Mutagenesis
[0099] Structure-guided site-specific mutagenesis represents a
powerful tool for the dissection and engineering of protein-ligand
interactions (Wells, 1996, Braisted et al., 1996). The technique
provides for the preparattion and testing of sequence variants by
introducing one or more nucleotide sequence changes into a selected
DNA.
[0100] 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.
[0101] 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. 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.
[0102] 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 multiresidue saturation mutagenesis are
daunting (Warren et al., 1996, Brown et al., 1996; Zeng et al.,
1996; Burton and Barbas, 1994; Yelton et al., 1995; Jackson et al.,
1995; Short et al., 1995; Wong et al., 1996; Hilton et al., 1996).
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. See also,
U.S. Pat. Nos. 5,798,208 and 5,830,650, for a description of
"walk-through" mutagenesis.
[0103] Other methods of site-directed mutagenesis are disclosed in
U.S. Pat. Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878;
5,389,514; 5,635,377; and 5,789,166.
[0104] In Vitro Scanning Mutagenesis
[0105] Random mutagenesis may be introduced using error prone PCR
(Cadwell and Joyce, 1992). The rate of mutagenesis may be increased
by performing PCR in multiple tubes with dilutions of
templates.
[0106] One particularly useful mutagenesis technique is alanine
scanning mutagenesis in which a number of residues are substituted
individually with the amino acid alanine so that the effects of
losing side-chain interactions can be determined, while minimizing
the risk of large-scale perturbations in protein conformation
(Cunningham et al., 1989).
[0107] In recent years, techniques for estimating the equilibrium
constant for ligand binding using minuscule amounts of protein have
been developed (Blackburn et al., 1991; U.S. Pat. Nos. 5,221,605
and 5,238,808). The ability to perform functional assays with small
amounts of material can be exploited to develop highly efficient,
in vitro methodologies for the saturation mutagenesis of
antibodies. The inventors bypassed cloning steps by combining PCR
mutagenesis with coupled in vitro transcription/translation for the
high throughput generation of protein mutants. Here, the PCR
products are used directly as the template for the in vitro
transcription/translation of the mutant single chain antibodies.
Because of the high efficiency with which all 19 amino acid
substitutions can be generated and analyzed in this way, it is now
possible to perform saturation mutagenesis on numerous residues of
interest, a process that can be described as in vitro scanning
saturation mutagenesis (Burks et al., 1997).
[0108] In vitro scanning saturation mutagenesis provides a rapid
method for obtaining a large amount of structure-function
information including: (i) identification of residues that modulate
ligand binding specificity, (ii) a better understanding of ligand
binding based on the identification of those amino acids that
retain activity and those that abolish activity at a given
location, (iii) an evaluation of the overall plasticity of an
active site or protein subdomain, (iv) identification of amino acid
substitutions that result in increased binding.
[0109] Dosage and Formulation
[0110] The composition (active ingredients; for example, SEQ ID
NO:1 or nucleotide sequence encoding it or a vector with nucleotide
sequence encoding SEQ ID NO:1) 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. A skilled
artisan is aware that a sufficient vector amount is utilized to
provide expression of a nucleotide sequence encoding SEQ ID NO:1 to
therapeutically effective levels. One skilled in the art recognizes
that the terms "administered" and "introduced" can be used
interchangeably.
[0111] The composition 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. They can be administered alone, but
are generally 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 and diabetes resulting from abnormalities in growth
hormone production. Furthermore they can also be used to stimulate
the growth or enhance feed efficiency of animals raised for meat
production, to enhance milk production, and stimulate egg
production.
[0112] The dosage administered will be 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; 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 the condition being
treated. The skilled worker 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
growth-related disorders will necessitate varying dosages from
individual to individual depending upon the degree of insufficiency
of growth hormone production. The dosage employed to stimulate
growth activity in livestock will be significantly higher (per kg
of subject weight) than the dosages employed to restore normal
growth in cases of growth hormone deficiencies such as pituitary
dwarfism in humans.
[0113] Thus, there is provided in accordance with this invention a
method of treating growth-related disorders characterized by
insufficient production of growth hormone which comprises
administering an amount of the analog of this invention sufficient
to stimulate the production of growth hormone to levels 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.
[0114] There is also provided a method of increasing the growth
rate of animals by administering an amount of the inventive GHRH
analog sufficient to stimulate the production of growth hormone at
a level greater than that associated with normal growth.
[0115] Gene Therapy Administration: Where appropriate, the gene
therapy 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.
[0116] 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.
[0117] 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.
[0118] 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).
[0119] 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.
[0120] 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 also 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.
[0121] The following examples are offered by way of example, and
are not intended to limit the scope of the invention in any
manner.
EXAMPLE 1
GHRH Super-Active Analogs Increase GH Secretagogue Activity and
Stability
[0122] 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. GHRH mutants were generated by site directed
mutagenesis. Gly15 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 Ala 15 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 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). 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
(GHRH-HV (FIG. 1A); H1V2A15L27N28).
EXAMPLE 2
DNA Constructs
[0123] 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 newly described synthetic muscle promoter, SPc5-12,
which contains a proximal serum response element frodm skeletal
.alpha.-actin, multiple MEF-2 sites, multiple MEF-1 sites, and
TEF-1 binding sites (Li et al., 1999). A 228-bp fragment of pGHRH,
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 hGH 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).
[0124] The wild type and mutated porcine GHRH cDNAs were obtained
by site directed mutagenesis of human GHRH cDNA (SEQ ID NO:8)
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 SEQ ID NO:2: 5'-AGGCAGCAGGGAGAGAGGAAC-
CAAGAGCAAGGAGCATAATGACTGCAG-3'. The porcine HV mutations were made
with the primer of SEQ ID NO:3:
5'-ACCCTCAGGATGCGGCGGCACGTAGATGCCATCTTCACCAAC-- 3'. The porcine
15Ala mutation was made with the primer of SEQ ID NO:4:
5'-CGGAAGGTGCTGGCCCAGCTGTCCGCC-3'. The porcine 27Leu28Asn mutation
was made with the primer of SEQ ID NO:5:
5'-CTGCTCCCAGGACATCCTGAACAGGCAGCAGGG- AGAG-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.
[0125] A skilled artisan is aware that instead of SEQ ID NO:8,
other GHRH sequences maybe utilized, including those from Mus
musculus (SEQ ID NO:9; GenBank Accession Number NM.sub.--010285);
Bos taurus (SEQ ID NO:10; GenBank Accession Number AF168686 or SEQ
ID NO:11; GenBank Accession Number BTU29611); Equus caballus (SEQ
ID NO:12; GenBank Accession Number AF097587); Rattus norvegicus
(SEQ ID NO:13; GenBank Accession Number RNU10156).
EXAMPLE 3
Cell Culture and Transfection
[0126] Experiments were performed in both pig anterior pituitary
culture and primary chicken myoblast cultures with equal success.
However, all figures demonstrate data generated with pig anterior
pituitary cultures. Primary chicken myoblast cultures were obtained
as follows. Chicken embronic tissue was harvested, dissected free
of skin and cartilage and mechanically dissociated. The cell
suspension was passed through cheesecloth and lens paper and plated
at a density of 1.times.10.sup.8 to 2.times.10.sup.8/100 mm plastic
culture dish. The cell populations which remained in suspension
were plated at a density of 2.times.10.sup.6 to 3.times.10.sup.6
cells/collagen-coated 100 mm plastic dish and incubated at
37.degree. C. in a 5% CO.sub.2 environment. Cells were then
incubated 24 hours prior to transfection at a density of
1.5.times.10.sup.6/100 mm plate in Minimal Essential Medium (MEM)
supplemented with 10% Heat Inactivated Horse Serum (HIHS), 5%
chicken embryo extract (CEE) (Gibco BRL; Grand Island, N.Y.), and
gentamycin. For further details see Draghia-Akli et al., 1997 and
Bergsma et al., 1986. The pig anterior pituitary culture was
obtained essentially as described (Tanner et al., 1990). Briefly,
pituitary tissue was dissociated under enzymatic conditions, plated
on plastic dishes for enough time to allow attachment. The cells
were then rinsed and exposed to incubation media prior to
experiments. For details see Tanner et al. (1990).
[0127] Cells were transfected with 4 mg of plasmid per 100 mm
plate, using lipofectamine, according to the manufacturer
instructions. After transfection, the medium was changed to MEM
which contained 2% HIHS and 2% CEE to allow the cells to
differentiate. Media and cells were harvested 72 hours
post-differentiation. The efficiency of transfection was estimated
by .beta.-galactosidase histochemistry of control plates to be 10%.
One day before harvesting, cells were washed twice in Hank's
Balanced Salt Solution (HBSS) and the media changed to MEM, 0.1%
bovine serum albumin. Conditioned media was treated by adding 0.25
volume of 1% trifluoroacetic acid and 1 mM
phenylmethylsulfonylflouride, frozen at -80.degree. C.,
lyophilized, purified on C-18 Sep-Columns (Peninsula Laboratories,
Belmont, Calif.), relyophilized and used in radioimmunoassays or
resuspended in media conditioned for primary pig anterior pituitary
culture.
EXAMPLE 4
GHRH Super-Active Analogs Increase GH Secretagogue Activity and
Stability
[0128] Skeletal myoblasts were transfected as in Example 3 with
each construct and GHRH moieties purified from conditioned culture
media cells were assayed for growth hormone secretion in pig
anterior pituitary cell cultures. As shown in FIG. 1B, media
collected after 24 hours and quantitated by porcine specific
GH-radioimmunoassays showed that modest gains in GH secretion
amounting to about 20% to 50% for the modified GHRH species
(GH15/27/28; GHRH-TI; GHRH-TV) over wild-type pGHRH. Only one of
the four mutants, GHRH-HV, had a substantial increase in GH
secretagogue activity in which pGH levels rose from baseline values
of 200 ng/ml up to 1600 ng/ml (FIG. 1B).
EXAMPLE 5
Plasma Incubation of HV-GHRH Molecule
[0129] Pooled porcine plasma was collected from control pigs, and
stored at -80.degree. C. Chemically synthesized HV-GHRH was
prepared by peptide synthesis. The porcine plasma was thawed and
centrifuged, placed at 37.degree. C. and allowed to equilibrate.
GHRH mutant was dissolved into plasma sample to a final
concentration of 100 .mu.g/ml. Immediately after the addition of
the GHRH mutant, and 15, 30, 60, 120 and 240 minutes later, 1 ml of
plasma was withdrawn and acidified with 1 ml of IM TFA. Acidified
plasma was purified on C18 affinity SEP-Pak columns, lyophilized
and analyzed by HPLC, using a Walters 600 multi-system delivery
system, a Walters intelligent sample processor, type 717 and a
Walters spectromonitor 490 (Walters Associates, Millipore Corp.,
Milford, Mass.). The detection was performed at 214 nm. The percent
of peptide degraded at these time points was measured by integrated
peak measurements.
[0130] Stability of wild type GHRH and the analog GHRH-HV was then
tested in porcine plasma, by incubation of GHRH peptides, followed
by solid phase extraction, and HPLC, analysis. As shown in FIG. 1C,
95% of the wild type GHRH (1-44)NH2 was degraded within 60 minutes
of incubation in plasma. In contrast, incubation of GHRH-HV in pig
plasma showed that at least 75% of the polypeptides was protected
against enzymatic cleavage, during 4 to 6 hours of incubation.
Thus, under identical conditions, a major portion of GHRH-HV
remained intact, while the wild-type GHRH is completely degraded,
indicating a considerable increase in stability for GHRH-HV to
serum proteases (FIG. 1C).
EXAMPLE 6
Animal Studies
[0131] Three groups of five, 3-4 weeks old hybrid cross barrows
(Yorkshire, Landrace, Hampshire and Duroc) were used in the GHRH
studies. The animals were individually housed with ad lib access to
water, and 6% of their body weight diet (24% protein pig meal,
Producers Cooperative Association, Bryan, Tex.). The animals were
weighed every other day, at 8:30 am, and the feed was subsequently
added. Animals were maintained in accordance with NIH Guide, USDA
and Animal Welfare Act guidelines.
EXAMPLE 7
Intramuscular Injection of Plasmid DNA in Porcine
[0132] Endotoxin-free plasmid (Qiagen Inc., Chatsworth, Calif.)
preparations of pSPc5-12-HV-GHRH, pSPc5-12-wt-GHRH and pSPc5-12bgal
were diluted in PBS (pH 7.4) to 1 mg/ml. The animals were assigned
equally to one of the treatments. The pigs were anesthetized with
isoflurane (concentration of 2-6% for induction and 1-3% for
maintenance). Jugular catheters were implanted by surgical
procedure to draw blood from the animals at day 3, 7, 14, 21, 28,
45 and 65 post-injection. While anesthetized, 10 mg of plasmid was
injected directly into the semitendinosus muscle of pigs. Two
minutes after injection, the injected muscle was placed in between
a set of calipers and electroporated using optimized conditions of
200V/cm with 4 pulses of 60 milliseconds (Aihara et al., 1998). At
65 days post-injection, animals were killed and internal organs and
injected muscle collected, weighed, frozen in liquid nitrogen, and
stored at -80.degree. C. Carcass' were weighed and analyzed by
neutron activation. Back fat was measured.
EXAMPLE 8
Muscle Injection of pSP-HV-GHRH Increases Porcine GHRH; GH and
IGF-I Serum Levels Over Two Months
[0133] The ability of the optimized protease resistant pSP-HV-GHRH
vector to facilitate long term expression of GHRH and stimulate GH
and IGF-I secreted levels was determined. Schematic maps of
pSP-HV-GHRH, as well as the wild-type construct, pSP-wt-GHRH, as a
wild-type control, and an synthetic myogenic promoter E. coli.
.beta.-galactosidase expression vector, pSP-bgal, as the placebo
control, is shown in FIG. 2A. Three-week-old castrated male-pigs
were anesthetized and a jugular vein catheter was inserted to allow
collection of blood samples with no discomfort for the animals.
Plasmid expression vector DNA (10 mg of DNA of pGHRH-HV; pSP-GHRH;
or pSP-bgal) was injected directly into semitendinosus muscle,
which was then electroporated (See Example 7).
EXAMPLE 9
Porcine GHRH, GH and IGF-1 Measurements
[0134] Porcine GHRH was measured by a heterologous human assay
system (Peninsula Laboratories, Belmont, Calif.). Sensitivity of
the assay is 1 pg/tube. Porcine GH in plasma was measured with a
specific double antibody procedure RIA (The Pennsylvania State
University). The sensitivity of the assay is 4 ng/tube. Porcine
IGF-1 was measured by heterologous human assay (Diagnostic System
Lab., Webster, Tex.). Data are analyzed using Microsoft Excel
statistics analysis package. Values shown in the figures are the
mean.+-.s.e.m. Specific p values were obtained by comparison using
Students t test. A p<0.05 is set as the level of statistical
significance. In pigs injected in semitendinosus muscle with
pSP-GHRH-HV, GHRH levels was increased at 7 days post-injection
(FIG. 2B), and were 150% above the control levels at 14 days
(652.4.+-.77 pg/ml versus 419.6.+-.13 pg/ml). pSP-GHRH-HV
expression activity reached a plateau by 60 days that was about 2
to 3 fold greater levels than the placebo injected control values.
The absolute quantity of serum GHRH, corrected for increased body
weight between day 0 and day 60 (blood volume accounts for 8% of
total body weight), secreted by the pSP-GHRH-HV injected pigs was 3
times greater than the placebo injected control values
(1426.49.+-.10.47 ng versus 266.84.+-.25.45 ng) (FIG. 2C). The
wild-type pSP-GHRH injected animals, which had been injected in
semitendinosus muscle, showed only a modest increase in their GHRH
levels starting with 45 days post-injection, but a 2-fold increase
by 60 days post-injection (779.36 ng), at levels sufficient to
elicit a biological effect.
[0135] Young animals have very high levels of GH that gradually
decrease with age. Blood samples, taken every 15 minutes over a
24-hour period after the 7 and 14 days following the initial
injections, were assayed for pGH levels which were extrapolated for
the total change in pGH content. The pGHRH-HV injected pigs (FIG.
2D) showed an increase in their GH content evident at day 7
post-injection (delta variation HV=+1.52, wt=-0.73 versus
control=-3.2 ng/ml) and 14 days post-injection (delta variation
HV=+1.09, wt=-4.42 versus control=-6.88 ng/ml).
[0136] Another indication of increased systemic levels of GH would
be elevated levels of IGF-I. Serum porcine IGF-1 levels started to
rise in pSP-GHRH-HV injected pigs at about 3 days post-injection
(FIG. 2E). At 21 days, these animals averaged about a 3-fold
increase in serum IGF-I levels, which was maintained over 60 days
(p<0.03). In comparison, pigs injected with the wild-type
pSP-GHRH expression vector had only a 40% increase in their
circulating IGF-1 levels (p=0.39), as shown in FIG. 2E.
EXAMPLE 10
Myogenic GHRH Expression Vectors Enhance Pig Growth
[0137] Porcine GH secreted into the systemic circulation after
intramuscular injection of myogenic pSP-GHRH expression vectors
augments growth over 65 days in castrated young male pigs. Body
composition measurements were performed either in vivo, at day 30
and 65 post-injection (densitometry, K40) or post-mortem (organ,
carcass, body fat, direct dissection followed by neutron activation
chamber). Wild-type pSP-GHRH injected animals were on average 21.5%
heavier than the placebo controls (37.125 kg vs. 29.375 kg), while
the pSP-GHRH-HV injected pigs were 37.8% heavier (41.775 kg;
p=0.014), as shown in FIG. 3A. Feed efficiency was also improved by
20% in pigs injected with GHRH constructs when compared with
controls (0.267 kg of food/day for each kg weight gain in
pSP-HV-GHRH, and 0.274 kg in pSP-wt-GHRH, versus 0.334 kg in
pSP-bgal injected pigs (FIG. 3B). Body composition studies by
densitometry, K40 potassium chamber and neutron activation chamber
showed a proportional increase of all body components in GHRH
injected animals, with no signs of organomegaly, relative
proportion of body fat and associated pathology. A photograph of a
placebo injected control pig and a pSP-GHRH-HV injected pig after
45 days is shown in FIG. 3C.
[0138] The metabolic profile of pSP-HV-GHRH injected pigs shown in
Table I connotes a significant decrease in serum urea level,
pSP-GHRH and pSP-GHRH-HV, respectively (9.+-.0.9 mg/dl in controls,
8.3.+-.1 mg/dl and 6.875.+-.0.5 mg/dl in injected pigs)(p=0.006),
indicating decreased amino acid catabolism. Serum glucose level was
similar between the controls and the plasmid GHRH injected pigs
(99.2.+-.4.8 mg/dl in control pigs, 104.8.+-.6.9 mg/dl in
pSP-GHRH-HV injected pigs and 97.5.+-.8 mg/dl in wildtype pSP-GHRH
injected animals (p=0.263). No other metabolic changes were
found.
1TABLE 1 The metabolic profile of GHRH injected pigs and controls
(values in mg/ml). total glucose urea creatinine protein Control
99.2 .+-. 4.8 9 .+-. 0.9 0.82 .+-. 0.06 4.6 .+-. 0.22 pSP-wt-GHRH
97.5 .+-. 8 8.3 .+-. 1 0.83 .+-. 0.056 4.76 .+-. 0.35 pSP-HV- 104.8
.+-. 6.9 6.875 .+-. 0.5 0.78 .+-. 0.04 4.88 .+-. 0.23 GHRH
EXAMPLE 11
Experiments with Different Levels of pSP-HV-GHRH
[0139] To further investigate the effects of pSP-HV-GHRH on the
growth in piglets, groups of 2 piglets were injected at 10 days
after birth with pSP-HV-GHRH (3 mg, 1 mg, 100 microg). As shown in
FIG. 4, the group injected with 100 micrograms of the plasmid
presented the best growth curve, with significantly statistically
differences to controls after 50 days of age. One animal in the
group injected with 3 mg developed antibodies and showed a
significantly decreased growth pattern.
[0140] Also, groups of 2 piglets were injected with the indicated
doses of pSP-HV-GHRH 10 days after birth. IGF-I values started to
rise 10 days post-injection, and at 35 days post-injection pigs
injected with 100 micrograms plasmid averaged 10.62 fold higher
IGF-I than the controls. Pigs injected with 1 mg averaged 7.94 fold
over the controls, and pigs injected with 3 mg averaged 1.16 fold
over control values.
[0141] Thus, in a specific embodiment lower dosages of pSP-HV-GHRH
are injected. In a specific embodiment about 100 micrograms (0.1
milligrams) of the plasmid is utilized. In another specific
embodiment about 200-300 micrograms are injected.
EXAMPLE 12
Age Comparisons with pSP-HV-GHRH
[0142] To optimize the age of piglets for pSP-HV-GHRH injection,
groups of 2 piglets were injected starting at birth with 2 mg
pSP-HV-GHRH. As shown in FIG. 6, the group injected 14 days after
birth presented the best growth curve, with significantly
statistically differences compared to the control at every time
point. One animal in the group injected at 21 days developed
antibodies and showed a significantly decreased growth pattern. It
is possible that there is insulin resistance if treated too early
(i.e. <about 10-14 days of age). In a specific embodiment the
therapy is most effective when natural GH and IGF-I levels are the
lowest (about 10-14 days of life), and may be counterproductive
when GHRH levels are normally high.
EXAMPLE 13
Summary
[0143] In summary, an optimal time point for injection is 14 days
after birth (an average 8 pounds heavier than the controls
(p<0.04) at 40 days post-injection). A preferred dosage for
injection is 100 micrograms plasmid in 2-5 ml volume (an average 6
pounds heavier than the controls (p<0.02) at 40 days
post-injection). Hormonal and biochemical constants are normal
(IGF-I, IGF-BP3, insulin, urea, glucose, total proteins,
creatinine) in the offspring of sow 1 (time course) and sow 3 (dose
curve) and in correlation with weight increase, with no deleterious
side effects. Body composition studies from the previous experiment
showed that HV-GHRH determined a uniform increase of all body
compartments (body composition similar to the controls but bigger),
while wt-GHRH determined an increase in lean body mass and a
decrease in fat.
[0144] All patents and publications mentioned in the specifications
are indicative of the levels of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
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[0214] One skilled in the art readily appreciates that the patent
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
14 1 40 PRT Artificial sequence Hormone 1 His Val Asp Ala Ile Phe
Thr Asn Ser Tyr Arg Lys Val Leu Ala Gln 1 5 10 15 Leu Ser Ala Arg
Lys Leu Leu Gln Asp Ile Leu Asn Arg Gln Gln Gly 20 25 30 Glu Arg
Asn Gln Glu Gln Gly Ala 35 40 2 48 DNA Artificial sequence Primer 2
aggcagcagg gagagaggaa ccaagagcaa ggagcataat gactgcag 48 3 42 DNA
Artificial sequence Primer 3 accctcagga tgcggcggca cgtagatgcc
atcttcacca ac 42 4 27 DNA Artificial sequence Primer 4 cggaaggtgc
tggcccagct gtccgcc 27 5 36 DNA Artificial sequence Primer 5
ctgctccagg acatcctgaa caggcagcag ggagag 36 6 358 DNA Artificial
sequence Promoter 6 gagctccacc gcggtggcgg ccgtccgcct tcggcaccat
cctcacgaca cccaaatatg 60 gcgacgggtg aggaatggtg gggagttatt
tttagagcgg tgaggaaggt gggcaggcag 120 caggtgttgg cgctttaaaa
ataactcccg ggagttattt ttagagcgga ggaatggtgg 180 acacccaaat
atggcgacgg ttcctcaccc gtcgccatat ttgggtgtcc gccctcggcc 240
ggggccgcat tcctgggggc cgggcggtgc tcccgcccgc ctcgataaaa ggctccgggg
300 ccggcggcgg cccacgagct acccggagga gcgggaggcg ccaagctcta gaactagt
358 7 649 DNA HUMAN 7 atcggggtgg catccctgtg acccctcccc agtgcctctc
ctggccctgg aagttgccac 60 tccagtgccc accagccttg tcctaataaa
attaagttgc atcattttgt ctgactaggt 120 gtccttctat aatattatgg
ggtggagggg ggtggtatgg agcaaggggc aagttgggaa 180 gacaacctgt
agggcctgcg gggtctattg ggaaccaagc tggagtgcag tggcacaatc 240
ttggctcact gcaatctccg cctcctgggt tcaagcgatt ctcctgcctc agcctcccga
300 gttgttggga ttccaggcat gcatgaccag gctcagctaa tttttgtttt
tttggtagag 360 acggggtttc accatattgg ccaggctggt ctccaactcc
taatctcagg tgatctaccc 420 accttggcct cccaaattgc tgggattaca
ggcgtgaacc actgctccct tccctgtcct 480 tctgatttta aaataactat
accagcagga ggacgtccag acacagcata ggctacctgc 540 catggcccaa
ccggtgggac atttgagttg cttgcttggc actgtcctct catgcgttgg 600
gtccactcag tagatgcctg ttgaattcaa gcttatcgat accgtcgac 649 8 262 DNA
HUMAN 8 atggtgctct gggtgttctt ctttgtgatc ctcaccctca gcaacagctc
ccactgctcc 60 ccacctcccc ctttgaccct caggatgcgg cggtatgcag
atgccatctt caccaacagc 120 taccggaagg tgctgggcca gctgtccgcc
cgcaagctgc tccaggacat catgagcagg 180 cagcagggag agagcaacca
agagcgagga gcgaggagca agggcacggc tttaatgact 240 gcaggaattc
gatatcaagc tt 262 9 632 DNA MOUSE 9 acccttatct ttccatcatt
tctttttcta acagcaaaga tcacaatgac agaagtgaat 60 gatcagaatg
taaaaatatt tgtgcaaaat tgcattaact gttctcacca tctaatcggg 120
gtacaacctc aaacacaacg gccataatga agaaaagcta cactggaagt tctagatgtc
180 atctggctcc cacaacatca cagagtccca cccaggagtg aaggatgctg
ctctgggtgc 240 tctttgtgat cctcatcctc accagtggct cccactgctc
actgcccccc tcacctccct 300 tcaggatgca gcgacacgta gatgccatct
tcaccaccaa ctacaggaaa ctcctgagcc 360 agctgtatgc ccggaaagtg
atccaggaca tcatgaacaa gcaaggggag aggatccagg 420 aacaaagggc
caggctcagc cgccaggaag acagcatgtg gacagaggac aagcagatga 480
ccctggagag catcttgcag ggattcccaa ggatgaagcc ttcagcggac gcttgagccc
540 cccgagcccc aaacacaact gtaccctgtt acttctgctt cagctctgac
cttttccgtc 600 ctctgtaaat acaataaaac ccccattctc at 632 10 391 DNA
BOS TAURUS 10 ctcaccctca gcagcggctc ccacggttcc ctgccttccc
agcctctcag gtaagcagtt 60 ctgagaagag aagcaagaga ggccctttga
ggatgcagac tcgagctggt ccccagctgg 120 gtcctcaggc agcctccctt
gctcatctct gggagggtgg cagactgagc cccagagagg 180 tcaccaccca
gccctggttc cagccctctc tggggacgag cagggcaaga ggcgacagaa 240
agacctcaca gagaccaagt gagcacagtc ccctgggcct cccaccccac cctttgacct
300 ctgactcctt ctactaggat tccacggtac gcagatgcca tcttcactaa
cagctaccgg 360 aaggttctgg gccagctgtc tgcccgcaac t 391 11 392 DNA
BOS TAURUS 11 ctcaccctca gcagcggctc ccacgggttc cctgccttcc
caagcctctc aggtaagcag 60 ttctgagaag agaagcaaga gaggcccttt
gaggatgcga ctcgagctgg tccccagctg 120 ggtcctcagg cagcctccct
tgctcatctc tgggagggtg gcagactgag ccccagagag 180 gtcaccaccc
agccctggtt ccagccctct ctggggacga gcagggcaag aggcgacaga 240
aagacctcac agagaccaag tgagcacagt cccctgggcc tcccacccca ccctttgacc
300 tctgactcct tctactagga ttccacggta cgcagatgcc atcttcacta
acagctaccg 360 gaaggttctg ggccagctgt ctgcccgcaa ct 392 12 88 DNA
EQUUS CABALLUS 12 atgcagatgc catcttcacc aacaactacc ggaaggtgct
gggccagctc tctgcccgca 60 agatcctcca ggacatcatg agcaggca 88 13 511
DNA RATTUS NORVEGICUS 13 ctgcggatgc cacggaacat cgagccaaat
cccaggaaca cgctctgaac cccaggagct 60 gcacaccact ctattaggtc
ccgcccagga gtgaaggatg ccactctggg tgttctttgt 120 gctcctcacc
ctcaccagtg gctcccactg ctcactgccc ccctcacctc ccttcagggt 180
gcggcggcat gcagacgcca tcttcaccag cagctaccgg agaatcctgg gccaattata
240 tgcccgcaaa ctgctgcacg aaatcatgaa caggcagcaa ggggagagga
accaggaaca 300 aagatccagg ttcaaccgcc atttggacag agtgtgggca
gaggacaagc agatggccct 360 ggagagcatc ttgcagggat tcccaaggat
gaagctttca gcggaggctt gagccctcgg 420 cccccaaaca tagctggacc
ctgttacttc tacttcagtt ctgatcttct ccttcctctg 480 tgaatacaat
aaagacccag ttctcatctg c 511 14 40 PRT PIG 14 Tyr Ala Asp Ala Ile
Phe Thr Asn Ser Tyr Arg Lys Val Leu Gly Gln 1 5 10 15 Leu Ser Ala
Arg Lys Leu Leu Gln Asp Ile Met Ser Arg Gln Gln Gly 20 25 30 Glu
Arg Asn Gln Glu Gln Gly Ala 35 40
* * * * *
References