U.S. patent application number 12/018582 was filed with the patent office on 2009-11-26 for composition and method to alter lean body mass and bone properties in a subject.
This patent application is currently assigned to VGX PHARMACEUTICLAS, INC.. Invention is credited to Ruxandra Draghia-Akli, Robert J. Schwartz.
Application Number | 20090292107 12/018582 |
Document ID | / |
Family ID | 23407110 |
Filed Date | 2009-11-26 |
United States Patent
Application |
20090292107 |
Kind Code |
A1 |
Draghia-Akli; Ruxandra ; et
al. |
November 26, 2009 |
COMPOSITION AND METHOD TO ALTER LEAN BODY MASS AND BONE PROPERTIES
IN A SUBJECT
Abstract
The present invention pertains to a method for decreasing the
body fat proportion, increasing lean body mass ("LBM"), increasing
bone density, or improving the rate of bone healing, or all, of a
subject. Overall, the embodiments of the invention can be
accomplished by delivering a heterologous nucleic acid sequence
encoding GHRH or functional biological equivalent thereof into the
cells of the subject and allowing expression of the encoded gene to
occur while the modified cells are within the subject. For
instance, when such a nucleic acid sequence is delivered into the
specific cells of the subject tissue specific constitutive
expression is achieved. Furthermore, external regulation of the
GHRH or functional biological equivalent thereof gene can be
accomplished by utilizing inducible promoters that are regulated by
molecular switch molecules, which are given to the subject. The
preferred method to deliver the constitutive or inducible nucleic
acid encoding sequences of GHRH or the functional biological
equivalents thereof is directly into the cells of the subject by
the process of in vivo electroporation. A decrease the body fat
proportion and an increase in lean body mass ("LBM"), or both of a
subject is achieved by the delivery of GHRH or functional
biological equivalent thereof as described herein by into the
subject as recombinant proteins. In addition, an increase in bone
density and improvement in the rate of bone healing is also
achieved.
Inventors: |
Draghia-Akli; Ruxandra;
(Houston, TX) ; Schwartz; Robert J.; (Houston,
TX) |
Correspondence
Address: |
Pepper Hamilton LLP
400 Berwyn Park, 899 Cassatt Road
Berwyn
PA
19312-1183
US
|
Assignee: |
VGX PHARMACEUTICLAS, INC.
BLUE BELL
PA
|
Family ID: |
23407110 |
Appl. No.: |
12/018582 |
Filed: |
January 23, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10281067 |
Oct 25, 2002 |
7338656 |
|
|
12018582 |
|
|
|
|
60357808 |
Oct 26, 2001 |
|
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Current U.S.
Class: |
530/324 |
Current CPC
Class: |
A61K 48/0083 20130101;
A61K 48/00 20130101; A61K 38/25 20130101; A61K 48/0041 20130101;
C07K 14/60 20130101; A61P 19/00 20180101; A61P 3/04 20180101; A61P
3/00 20180101; A61K 48/005 20130101 |
Class at
Publication: |
530/324 |
International
Class: |
C07K 14/00 20060101
C07K014/00 |
Claims
1-167. (canceled)
168. A peptide for altering bone density comprising a functional
biological equivalent of growth hormone releasing hormone ("GHRH"),
wherein the GHRH is a polypeptide that is biologically active in a
subject; and the functional biological equivalent of GHRH is a
polypeptide that has been engineered to contain distinct amino acid
sequences while simultaneously having similar or improved
biologically activity when compared to GHRH.
169. The peptide of claim 168 comprising the formula:
-X.sub.1-X.sub.2-DAIFTNSYRKVL-X.sub.3-QLSARKLLQDI-X.sub.4-X.sub.5-RQQGERN-
QEQGA-OH wherein the formula has the following characteristics:
X.sub.1 is a D- or L-isomer of the amino acid tyrosine ("Y"), or
histidine ("H"); X.sub.2 is a D- or L-isomer of the amino acid
alanine ("A"), valine ("V"), or isoleucine ("I"); X.sub.3 is a D-
or L-isomer of the amino acid alanine ("A") or glycine ("G");
X.sub.4 is a D- or L-isomer of the amino acid methionine ("M"), or
leucine ("L"); X.sub.5 is a D- or L-isomer of the amino acid serine
("S") or asparagine ("N"); or a combination thereof.
170. The peptide of claim 168 comprising SeqID No: 1; SeqID No: 2;
SeqID No: 3; or SeqID No: 4.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application Ser. No. 60/357,808 entitled "increase Body mass,
decrease body fat proportion, increase bone density and improve
bone healing rate," filed on Oct. 26, 2001, the entire content of
which is hereby incorporated by reference.
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BACKGROUND
[0155] The present invention pertains to compositions and methods
for plasmid-mediated gene supplementation. The present invention
relates to a method of decreasing body fat proportions and
increasing lean body mass ("LBM") in an animal subject. Overall,
the embodiments of the invention can be accomplished by delivering
a nucleic acid expression construct that encodes a GHRH or
functional biological equivalent thereof into a tissue of a subject
and allowing expression of the encoded gene in the subject. For
example, when such a nucleic acid sequence is delivered into the
specific cells of the subject tissue specific constitutive
expression is achieved. Furthermore, external regulation of the
GHRH or functional biological equivalent thereof gene can be
accomplished by utilizing inducible promoters that are regulated by
molecular switch molecules, which are given to the subject. The
preferred method to deliver the constitutive or inducible nucleic
acid encoding sequences of GHRH or the functional biological
equivalents thereof is directly into the cells of the subject by
the process of in vivo electroporation. In addition, this invention
also relates to a method of increasing bone density and improvising
the rate of bone healing in an animal subject. More specifically,
this invention pertains to both an in vivo and an ex vivo method
for delivering a heterologous nucleic acid sequence encoding growth
hormone releasing hormone "GHRH" or functional biological
equivalent thereof into the cells of the subject and allowing
expression of the encoded gene to occur while the modified cells
are within the subject. Another embodiment of the present invention
relates to regulating the expression of the GHRH using a molecular
switch (e.g. mifepistone).
[0156] Growth Hormone ("GH") and Immune Function: The central role
of growth hormone ("GH") in controlling somatic growth in humans
and other vertebrates, and the physiologically relevant pathways
regulating GH secretion from the pituitary are well known. The GH
production pathway is composed of a series of interdependent genes
whose products are required for normal growth. The GH pathway genes
include: (1) ligands, such as GH and insulin-like growth factor-I
("IGF-I"); (2) transcription factors such as prophet of pit 1, or
prop 1, and pit 1: (3) agonists and antagonists, such as growth
hormone releasing hormone ("GHRH") and somatostatin ("SS"),
respectively; and (4) receptors, such as GHRH receptor ("GHRH-R")
and the GH receptor ("GH-R"). These genes are expressed in
different organs and tissues, including 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.
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 elicits both
direct and indirect actions on peripheral tissues, the indirect
effects being mediated mainly by IGF-I.
[0157] 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, as well as non-GH-deficient
short stature. Non-GH-deficiencies have different etiology, such
as: (1) genetic diseases, Turner syndrome, hypochondroplasia; and
(2) chronic renal insufficiency. 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 and GH therapy has been shown to
be effective (Gesundheit and Alexander, 1995).
[0158] 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-I axis undergoes dramatic changes
through aging and in the elderly with decreased GH production rate
and GH half-life, decreased IGF-I response to GH and GHRH stimuli
leads to loss of skeletal muscle mass (sarcopenia), osteoporosis,
and increase in fat and decrease in lean body mass. Previous
studies have shown that in elderly the level of GH secretion is
significant reduced by 70-80% of teenage level. It has been
demonstrated that the development of sarcopenia can be offset by
exogenous GH therapy. However, this remains a controversial therapy
in the elderly because of its cost and frequent side effects.
[0159] 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, this therapy has several
disadvantages, including an impractical requirement for frequent
administration of GH and undesirable secondary effects.
[0160] GH is released in a distinctive pulsatile pattern that has
profound importance for its biological activity (Argente et al.,
1996). Secretion of GH is stimulated by the natural GH
secretagogue, GHRH, and inhibited by somatostatin (SS), and both
hypothalamic hormones (Thorner et al., 1990). GH pulses are a
result of GHRH secretion that is associated with a diminution or
withdrawal of somatostatin secretion. In addition, the pulse
generator mechanism is timed by GH-negative feedback. The
endogenous rhythm of GH secretion becomes entrained to the imposed
rhythm of exogenous GH administration. Effective and regulated
expression of the GH and insulin-like growth factor I ("IGF-I")
pathway is essential for optimal linear growth, homeostasis of
carbohydrate, protein, and fat metabolism, and for providing a
positive nitrogen balance (Murray and Shalet, 2000). Numerous
studies in humans, sheep or pigs showed that continuous infusion
with recombinant GHRH protein restores the normal GH pattern
without desensitizing GHRH receptors or depleting GH supplies as
this system is capable of feed-back regulation, which is abolished
in the GH therapies (Dubreuil et al., 1990a; Vance et al., 1985b;
Vance, 1990; Vance et al., 1985a). Thus, GHRH recombinant protein
treatment may be more physiologically relevant than GH therapy.
However, due to the short half-life of GHRH in vivo, frequent (one
to three times per day) intravenous, subcutaneous or intranasal
(requiring 300-fold higher dose) administrations are necessary
(Evans et al., 2001; Thorner et al., 1986). Thus, as a chronic
therapy, recombinant GHRH protein administration is not
practical.
[0161] 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 (Thorner et al., 1984). Administration of
recombinant GHRH to GH-deficient children or adult humans augments
IGF-I levels, increases GH secretion proportionally to the GHRH
dose, yet still invokes a response to bolus doses of GHRH (Bercu et
al., 1997). Thus, GHRH administration represents a more
physiological alternative of increasing subnormal GH and IGF-I
levels (Corpas et al., 1993).
[0162] 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. Extracranially secreted GHRH, as
processed protein species GHRH(1-40) hydroxy or GHRH(1-44) amide or
even as shorter truncated molecules, are biological active (Thorner
et al., 1984). It has been reported that a low level of GHRH (100
pg/ml) in the blood supply stimulates GH secretion (Corpas et al.,
1993). Direct plasmid DNA gene transfer is currently the basis of
many emerging therapy strategies and thus does not require viral
genes or lipid particles (Aihara and Miyazaki, 1998; Muramatsu et
al., 1998). Skeletal muscle is target tissue, because muscle fiber
has a long life span and can be transduced by circular DNA plasmids
that express over months or years in an immunocompetent host (Davis
et al., 1993; Tripathy et al., 1996). Previous reports demonstrated
that human GHRH cDNA could be delivered to muscle by an injectable
myogenic expression vector in mice where it transiently stimulated
GH secretion to a modes extent over a period of two weeks
(Draghia-Akli et al., 1997).
[0163] Wild type GHRH has a relatively short half-life in the
circulatory system, both in humans (Frohman et al., 1984) and in
farm animals. After 60 minutes of incubation in plasma 95% of the
GHRH(1-44)NH2 is degraded, while incubation of the shorter (1-40)OH
form of the hormone, under similar conditions, shows only a 77%
degradation of the peptide after 60 minutes of incubation (Frohman
et al., 1989). Incorporation of cDNA coding for a particular
protease-resistant GHRH analog in a therapeutic nucleic acid vector
results in a molecule with a longer half-life in serum, increased
potency, and provides greater GH release in plasmid-injected
animals (Draghia-Akli et al., 1999), herein incorporated by
reference). Mutagenesis via amino acid replacement of protease
sensitive amino acids prolongs the serum half-life of the GHRH
molecule. Furthermore, the enhancement of biological activity of
GHRH is achieved by using super-active analogs that may increase
its binding affinity to specific receptors (Draghia-Akli et al.,
1999).
[0164] Administering novel GHRH analog proteins (U.S. Pat. Nos.
5,847,066; 5,846,936; 5,792,747; 5,776,901; 5,696,089; 5,486,505;
5,137,872; 5,084,442, 5,036,045; 5,023,322; 4,839,344; 4,410,512,
RE33,699) or synthetic or naturally occurring peptide fragments of
GHRH (U.S. Pat. Nos. 4,833,166; 4,228,158; 4,228,156; 4,226,857;
4,224,316; 4,223,021; 4,223,020; 4,223,019) for the purpose of
increasing release of growth hormone have been reported. A GHRH
analog containing the following mutations 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 GHRH analog is the subject of U.S.
patent application Ser. No. 09/624,268 ("the '268 patent
application"), which teaches application of a GHRH analog
containing mutations that improve the ability to elicit the release
of growth hormone. In addition, the '268 patent application relates
to the treatment of growth deficiencies; the improvement of growth
performance; the stimulation of production of growth hormone in an
animal at a greater level than that associated with normal growth;
and the enhancement of growth utilizing the administration of
growth hormone releasing hormone analog and is herein incorporated
by reference. In the embodiments of the '268 patent application and
specific embodiments herein, the mutated GHRH-encoding molecules
lack the Gln, Ser or Thr mutations of the Asn at position 8.
[0165] U.S. Pat. No. 5,061,690 is directed toward increasing both
birth weight and milk production by supplying to pregnant female
mammals an effective amount of human GHRH or one of it analogs for
10-20 days. Application of the analogs lasts only throughout the
lactation period. However, multiple administrations are presented,
and there is no disclosure regarding administration of the growth
hormone releasing hormone (or factor) as a DNA molecule, such as
with plasmid mediated supplementation techniques.
[0166] U.S. Pat. Nos. 5,134,120 ("the '120 patent") and 5,292,721
("the '721 patent") teach that by deliberately increasing growth
hormone in swine during the last 2 weeks of pregnancy through a 3
week lactation resulted in the newborn piglets having marked
enhancement of the ability to maintain plasma concentrations of
glucose and free fatty acids when fasted after birth. In addition,
the '120 and '721 patents teaches that treatment of the sow during
lactation results in increased milk fat in the colostrum and an
increased milk yield. These effects are important in enhancing
survivability of newborn pigs and weight gain prior to weaning.
However the '120 and '721 patents provide no teachings regarding
administration of the growth hormone releasing hormone as a DNA
form.
[0167] In contrast to protein therapy, nucleic acid transfer
delivers polynucleotides to somatic tissue in a manner that, in
some embodiments, can correct inborn or acquired deficiencies and
imbalances. In other embodiments, vectors such as plasmids are used
to supplement basal levels of an expressed endogenous gene product.
Gene-based drug delivery offers a number of advantages over the
administration of recombinant proteins. These advantages include
the conservation of native protein structure, improved biological
activity, avoidance of systemic toxicities, and avoidance of
infectious and toxic impurities. In addition, nucleic acid
transfer, for therapeutic purposes, and plasmid-mediated
supplementation of an endogenous gene product allow for prolonged
exposure to the protein in the therapeutic range, because the newly
secreted protein is present continuously in the blood
circulation.
[0168] The primary limitation of using recombinant protein is the
limited availability of protein after each administration.
Plasmid-mediated gene supplementation using injectable DNA plasmid
vectors overcomes this, because a single injection into the
patient's skeletal muscle permits physiologic expression for
extensive periods of time (WO 99/05300 and WO 01/06988). Injection
of the vectors promotes the production of enzymes and hormones in
animals in a manner that more closely mimics the natural process.
Furthermore, among the non-viral techniques for gene transfer in
vivo, the direct injection of plasmid DNA into muscle tissue is
simple, inexpensive, and safe.
[0169] In a plasmid-mediated supplementation expression system, a
non-viral nucleic acid vector, such as a plasmid, may comprise a
synthetic nucleic acid delivery system in addition to a nucleic
acid encoding the GHRH being supplemented. In this way, the risks
associated with the use of most viral vectors can be avoided. The
non-viral expression vector products generally have low toxicity
due to the use of "species-specific" components for nucleic acid
delivery, which minimizes the risks of immunogenicity generally
associated with viral vectors. Additionally, no integration of
plasmid sequences into host chromosomes has been reported in vivo
to date, so that this type of plasmid-mediated supplementation of
GHRH, should neither activate oncogenes nor inactivate tumor
suppressor genes. As episomal systems residing outside the
chromosomes, plasmids have defined pharmacokinetics and elimination
profiles, leading to a finite duration of gene expression in target
tissues. Furthermore, the plasmid DNA could be engineered so it
would be delivered to the cells in a linear rather than circular
form (which would further prevent any genomic integration event);
the plasmid could be deleted of the antibiotic resistance gene and
bacterial origin of replication, making it completely safe for in
vivo therapy.
[0170] Efforts have been made to enhance the delivery of plasmid
DNA to cells by physical means including electroporation,
sonoporation, and pressure. Injection by electroporation involves
the application of a pulsed electric field to create transient
pores in the cellular membrane without causing permanent damage to
the cell. It thereby allows for the introduction of exogenous
molecules (Smith and Nordstrom, 2000). By adjusting the electrical
pulse generated by an electrophoretic system, nucleic acid
molecules can travel through passageways or pores in the cell that
are created during the procedure. U.S. Pat. No. 5,704,908 describes
an electroporation apparatus for delivering molecules to cells at a
selected location within a cavity in the body of a patient. These
pulse voltage injection devices are also described in U.S. Pat.
Nos. 5,439,440 and 5,702,304, and PCT WO 96/12520, 96/12006,
95/19805, and 97/07826.
[0171] Recently, significant progress has been obtained using
electroporation to enhance plasmid delivery in vivo.
Electroporation has been used very successfully to transfect tumor
cells after injection of plasmid (Lucas et al., 2002; Matsubara et
al., 2001) or to deliver the anti-tumor drug bleomycin to cutaneous
and subcutaneous tumors in humans (Gehl et al., 1998; Heller et
al., 1996). Electroporation also has been extensively used in mice
(Lesbordes et al., 2002; Lucas et al., 2001; Vilquin et al., 2001),
rats (Terada et al., 2001; Yasui et al., 2001), and dogs (Fewell et
al., 2001) to deliver therapeutic genes that encode for a variety
of hormones, cytokines or enzymes. Our previous studies using
growth hormone releasing hormone ("GHRH") showed that plasmid
therapy with electroporation is scalable and represents a promising
approach to induce production and regulated secretion of proteins
in large animals and humans (Draghia-Akli et al., 1999;
Draghia-Akli et al., 2002).
[0172] The ability of electroporation to enhance plasmid uptake
into the skeletal muscle has been well documented, as described
above. In addition, plasmid formulated with poly-L-glutamate
("PLG") or polyvinylpyrolidone (PVP) has been observed to increase
plasmid transfection and consequently expression of the desired
transgene. The anionic polymer sodium PLG could enhance plasmid
uptake at low plasmid concentrations, while reducing any possible
tissue damage caused by the procedure. The ability of
electroporation to enhance plasmid uptake into the skeletal muscle
has been well documented, as previously described. PLG is a stable
compound and resistant to relatively high temperatures (Dolnik et
al., 1993). PLG has been previously used to increase stability in
vaccine preparations (Matsuo et al., 1994) without increasing their
immunogenicity. It also has been used as an anti-toxin post-antigen
inhalation or exposure to ozone (Fryer and Jacoby, 1993). In
addition, plasmid formulated with PLG or polyvinylpyrrolidone (PVP)
has been observed to increase gene transfection and consequently
gene expression to up to 10 fold in the skeletal muscle of mice,
rats and dogs (Fewell et al., 2001; Mumper et al., 1998). PLG has
been used to increase stability of anti-cancer drugs (Li et al.,
2000) and as "glue" to close wounds or to prevent bleeding from
tissues during wound and tissue repair (Otani et al., 1996; Otani
et al., 1998).
[0173] Although not wanting to be bound by theory, PLG will
increase the transfection of the plasmid during the electroporation
process, not only by stabilizing the plasmid DNA, and facilitating
the intracellular transport through the membrane pores, but also
through an active mechanism. For example, positively charged
surface proteins on the cells could complex the negatively charged
PLG linked to plasmid DNA through protein-protein interactions.
When an electric field is applied, the surface proteins reverse
direction and actively internalize the DNA molecules, process that
substantially increases the transfection efficiency.
[0174] The use of directly injectable DNA plasmid vectors has been
limited in the past. The inefficient DNA uptake into muscle fibers
after simple direct injection has led to relatively low expression
levels (Prentice et al., 1994; Wells et al., 1997) In addition, the
duration of the transgene expression has been short (Wolff et al.,
1990). The most successful previous clinical applications have been
confined to vaccines (Danko and Wolff, 1994; Tsurumi et al.,
1996).
[0175] Although there are references in the art directed to
electroporation of eukaryotic cells with linear DNA (McNally et
al., 1988; Neumann et al., 1982) (Toneguzzo et al., 1988) (Aratani
et al., 1992; Nairn et al., 1993; Xie and Tsong, 1993; Yorifuji and
Mikawa, 1990), these examples illustrate transfection into cell
suspensions, cell cultures, and the like, and the transfected cells
are not present in a somatic tissue.
[0176] U.S. Pat. No. 4,956,288 is directed to methods for preparing
recombinant host cells containing high copy number of a foreign DNA
by electroporating a population of cells in the presence of the
foreign DNA, culturing the cells, and killing the cells having a
low copy number of the foreign DNA.
[0177] U.S. Pat. No. 5,874,534 ("the '534 patent") and U.S. Pat.
No. 5,935,934 ("the '934 patent") describe mutated steroid
receptors, methods for their use and a molecular switch for nucleic
acid transfer, the entire content of each is hereby incorporated by
reference. A molecular switch for regulating expression in nucleic
acid transfer and methods of employing the molecular switch in
humans, animals, transgenic animals and plants (e.g.
GeneSwitch.RTM.) are described in the '534 patent and the '934
patent. The molecular switch is described as a method for
regulating expression of a heterologous nucleic acid cassette for
nucleic acid transfer and is comprised of a modified steroid
receptor that includes a natural steroid receptor DNA binding
domain attached to a modified ligand binding domain. The modified
binding domain usually binds only non-natural ligands,
anti-hormones or non-native ligands. One skilled in the art readily
recognizes natural ligands do not readily bind the modified
ligand-binding domain and consequently have very little, if any,
influence on the regulation and/or expression of the gene contained
in the nucleic acid cassette.
[0178] Thus, the present invention is directed to a novel method of
increasing lean body mass, decreasing body fat proportions,
increasing bone density, and/or increasing the rate of bone healing
in an animal by plasmid-mediated supplementation of GHRH.
SUMMARY
[0179] One embodiment of the present invention pertains to a method
for decreasing the body fat proportion, increasing lean body mass
("LBM"), increasing bone density, and increasing the rate of bone
healing of a subject by utilizing a nucleic acid sequence
containing both a constitutive promoter and an encoding sequence
for growth hormone releasing hormone ("GHRH") or analog thereof.
When this nucleic acid sequence is delivered into the specific
cells of the subject (e.g. somatic cells, stem cells, or germ
cells), tissue specific constitutive expression of GHRH is
achieved. The preferred method to deliver the nucleic acid sequence
with the constitutive promoter and the encoding sequence of GHRH or
the analog thereof is directly into the cells of the subject by the
process of in vivo electroporation. Electroporation may involve
externally supplied electrodes, or in the case of needles,
internally supplied electrodes to aid in the inclusion of desired
nucleotide sequences into the cells of a subject while the cells
are within a tissue of the subject.
[0180] Another embodiment of the present invention pertains to a
method for decreasing the body fat proportion, increasing LBM,
increasing bone density, and increasing bone healing rate of a
subject by utilizing the ability to regulate the expression of GHRH
or analog thereof. Regulation is achieved by delivering into the
cells of the subject a first nucleic acid sequence, and a second
nucleic acid sequence, followed by a molecular switch; where the
first nucleic acid sequence contains an inducible-promoter with a
coding region for a growth-hormone-releasing-hormone
("inducible-GHRH") or an analog thereof and the second nucleic acid
sequence has a constitutive promoter with a coding region for an
inactive regulator protein. By delivering a molecular switch
molecule (e.g. mifepistone) into the subject, the inactive
regulator protein becomes active and initiates transcription of the
inducible-GHRH in the subject. The expression and ensuing release
of GHRH or analog thereof by the modified-cells within the subject
will decrease the body fat proportion and increase the LBM of the
subject in a manner that can be regulated by external molecular
switch molecules (e.g. mifepistone). The delivery of the nucleic
acid sequences that allow external regulation of GHRH or the analog
thereof directly into the cells of the subject can be accomplished
by the process of in vivo electroporation.
[0181] A further embodiment of the present invention pertains to a
method for increasing lean body mass, decreasing body fat
proportion, increasing bone density, increasing the rate of bone
healing, or a combination thereof, of a subject by utilizing
therapy that introduces specific recombinant GHRH-analog protein
into the subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0182] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0183] FIG. 1 shows the amino acid sequence of GHRH or functional
biological equivalent thereof. All mutant sequences were obtained
by site directed mutagenesis of the porcine wild type sequence.
[0184] FIG. 2 shows the body weight of SCID mice that were injected
with 7.5 micrograms of pSP-GHRH mutants.
[0185] FIG. 3 shows the body composition of SCID mice that were
injected with 7.5 micrograms of plasmid expressing either one of
the GHRH mutants or a pSP-beta-galactosidase as control.
[0186] FIG. 4 shows the bone area of SCID mice that were injected
with 7.5 micrograms of plasmid expressing either one of the GHRH
mutants or a pSP-beta-galactosidase as control.
[0187] FIG. 5 shows the IGF-I levels of SCID mice that were
injected with 7.5 micrograms of plasmid expressing either one of
the GHRH mutants or a pSP-beta-galactosidase as control.
[0188] FIG. 6 shows a schematic of the mifepristone-dependent
GHRH/GeneSwitch.RTM. system in primary myoblasts in vitro. Plasmid
structures and schematic for how the GeneSwitch.RTM. system works
are illustrated. Plasmid p1633 encodes for the GeneSwitch.RTM.
regulator protein, which is a chimera of yeast GAL4 DNA binding
domain ("GAL4"), truncated human progesterone receptor
ligand-binding domain ("hPR LBD"), and activation domain from the
p65 subunit of human NF-.quadrature.B ("p65"). The protein is
synthesized as an inactive monomer. Binding of mifepristone
triggers a conformational change that leads to activation and
dimerization. Activated homodimers bind to GAL4 sites in the
inducible promoter and stimulate transcription of the GHRH
gene.
[0189] FIG. 7 shows the function of a mifepristone-dependent
GHRH/GeneSwitch.RTM. system in primary myoblasts in vitro. Northern
blot analysis of inducible GHRH constructs. Primary chicken
myoblast cultures were obtained and transfected as described
previously (Bergsma et al., 1986; Draghia-Akli et al., 1997), with
4 micrograms of a 10:1 mixture of inducible GHRH ("pGR1774") and
GeneSwitch.RTM. plasmids ("pGS1633"). A Muscle specific synthetic
promoter (Li et al., 1999) driven construct coding for E. coli
beta-galactosidase, .quadrature.gal, is used as a negative control.
As a positive control, cells were transfected with a constitutively
active pSP-GHRH construct (Draghia-Akli et al., 1999). In the
figure, Nt=non-transfected cells; .quadrature.-gal=cells
transfected with pSP-.quadrature.-gal construct; SP-GHRH=cells
transfected with a constitutively active GHRH construct;
+MFP=mifepristone was added to the culture media; and
-MFP=mifepristone was not added to the culture media. Ethidium
bromide gels are included as loading controls.
[0190] FIG. 8 shows that the mifepristone dosing induces serum
IGF-I levels in SCID mice that received a single administration of
GHRH/GeneSwitch.RTM. plasmids. Values are presented as fold
activation over control levels. The area under the dark line
represents normal variability of IGF-I levels in adult animals. The
table contains the p values for the induced peaks. The p values C
v. A indicate comparison between animals injected with the
.quadrature.-gal construct versus animals injected with the IS+MFP;
C v. B. indicates comparison between animals injected with the IS
with and without the MFP.
[0191] FIG. 9 shows the enhanced weight gain during a chronic 149
day MFP induction. Average weight increased in injected mice upon
chronic activation of the GHRH/GeneSwitch.RTM. system
(*p<0.027).
[0192] FIG. 10 shows the increase in pituitary weight with a
chronic 149 day MFP induction. Pituitary weight/total body weight
in +MFP injected animals (*p<0.035).
[0193] FIG. 11 shows the improved body composition in chronically
induced GHRH/GeneSwitch.RTM. mice. Body composition measurements
were performed either under anesthesia, at day 149 post-injection
("PIXImus") or post-mortem (organ, carcass, body fat, direct
dissection of the body). Lean non-bone mass is significantly
increased (*p<0.022).
[0194] FIG. 12 shows the improved fat body mass/total weight in
chronically induced GHRH/GeneSwitch.RTM. mice. Fat body mass/total
weight measurements were performed either under anesthesia, at day
149 post-injection ("PIXImus") or post-mortem (organ, carcass, body
fat, direct dissection of the body). Fat body mass/total weight is
significantly decreased in induced animals (*p<0.05).
[0195] FIG. 13 shows the increased bone area in chronically induced
GHRH/GeneSwitch.RTM. mice. Bone area measurements were performed
either under anesthesia, at day 149 post-injection ("PIXImus") or
post-mortem (organ, carcass, body fat, direct dissection of the
body). Bone area is increased by PIXImus (*p<0.0006).
[0196] FIG. 14 shows the increased mineral content in chronically
induced GHRH/GeneSwitch.RTM. mice. Bone mineral content
measurements were performed either under anesthesia, at day 149
post-injection ("PIXImus") or post-mortem (organ, carcass, body
fat, direct dissection of the body). Bone mineral content is
increased in induced animals (*p<0.002).
[0197] FIG. 15 shows the secreted embryonic alkaline phosphatase
("SEAP") plasma concentration in pigs at 0 to 7 days
post-injection. Different needle-type electrodes were compared with
calipers electrodes following the plasmid injection into the
muscle. In the figure, N6=Six-needle array electrode: 21 gauge
needles, 2 cm length mounted on a 1 cm-diameter array;
N3=three-needle array device: two solid needles, 1 median
hypodermic needle, 21 gauge, 2 cm length; and C=caliper electrode:
2 solid square plate electrodes, 1.5 cm. Voltage and number of
pulses are also indicated. At 7 days post-injection p<0.006 for
N3/200V/6 pulses and N6/100V/6 pulses groups, and p<0.0035 for
N6/200V/6 pulses group.
[0198] FIG. 16 shows the body weights of pigs injected at 10 days
of age with 3, 1 and 0.1 mg of pSP-HV-GHRH or vehicle. The greatest
weight gain was achieved by pigs injected with the lowest dose,
with statistically significant differences from the controls at all
time points tested (p<0.02). Values are means.+-.s.e.m.
[0199] FIG. 17 shows the body weights of pigs injected with 2
milligrams of pSP-HV-GHRH at 0, 7, 14 and 21 days of age. Animal
injected at 14 days of age showed the greatest weight gain,
statistically different from the controls at all time points tested
(p<0.02). Values are means.+-.s.e.m.
[0200] FIG. 18 shows the plasma IGF-I concentration after direct
intramuscular injection of the different quantities of pSP-HV-GHRH
construct. Values are means.+-.s.e.m.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0201] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
DEFINITIONS
[0202] The term "a" or "an" as used herein in the specification may
mean one or more. As used herein in the claim(s), when used in
conjunction with the word "comprising", the words "a" or "an" may
mean one or more than one. As used herein "another" may mean at
least a second or more.
[0203] The term "any range derivable therein" as used herein means
a range selected from the numbers described in the specification,
and "any integer derivable therein" means any integer between such
a range.
[0204] The term "analog" as used herein includes any mutant of
GHRH, or synthetic or naturally occurring peptide fragments of
GHRH, such as HV-GHRH (SeqID No: 1), TI-GHRH (SeqID No: 2), TV-GHRH
(SeqID No: 3), 15/27/28-GHRH (SeqID No: 4), (1-44)NH.sub.2 (SeqID
No: 5) or (1-40)OH (SeqID No: 6) forms, or any shorter form to no
less than (1-29) amino acids.
[0205] The term "bone density" as used herein is defined as the
density of minerals in the bone as measured by a standard means in
the art, such as x-ray, MRI, dual-energy x-ray absorbitometry
(DEXA), or any advanced imaging system in the art.
[0206] The term "cassette" as used herein is defined as one or more
transgene expression vectors.
[0207] The term "cell-transfecting pulse" as used herein is defined
as a transmission of a force which results in transfection of a
vector, such as a linear DNA fragment, into a cell. In some
embodiments, the force is from electricity, as in electroporation,
or the force is from vascular pressure.
[0208] The term "coding region" as used herein refers to any
portion of the DNA sequence that is transcribed into messenger RNA
(mRNA) and then translated into a sequence of amino acids
characteristic of a specific polypeptide.
[0209] The term "delivery" or "delivering" as used herein is
defined as a means of introducing a material into a tissue, a
subject, a cell or any recipient, by means of chemical or
biological process, injection, mixing, electroporation,
sonoporation, or combination thereof, either under or without
pressure.
[0210] The term "donor-cells" as used herein refers to any cells
that have been removed and maintained in a viable state for any
period of time outside the donor-subject.
[0211] The term "donor-subject" as used herein refers to any
species of the animal kingdom wherein cells have been removed and
maintained in a viable state for any period of time outside the
subject.
[0212] The term "DNA fragment" or "nucleic acid expression
construct" as used herein refers to a substantially double stranded
DNA molecule. Although the fragment may be generated by any
standard molecular biology means known in the art, in some
embodiments the DNA fragment or expression construct is generated
by restriction digestion of a parent DNA molecule. The terms
"expression vector," "expression cassette," or "expression plasmid"
can also be used interchangeably. Although the parent molecule may
be any standard molecular biology DNA reagent, in some embodiments
the parent DNA molecule is a plasmid.
[0213] The term "electroporation" as used herein refers to a method
that utilizes electrical pulses to deliver a nucleic acid sequence
into cells.
[0214] The terms "electrical pulse" and "electroporation" as used
herein refer to the administration of an electrical current to a
tissue or cell for the purpose of taking up a nucleic acid molecule
into a cell. A skilled artisan recognizes that these terms are
associated with the terms "pulsed electric field" "pulsed current
device" and "pulse voltage device." A skilled artisan recognizes
that the amount and duration of the electrical pulse is dependent
on the tissue, size, and overall health of the recipient subject,
and furthermore knows how to determine such parameters
empirically.
[0215] The term "encoded GHRH" as used herein is a biologically
active polypeptide of GHRH.
[0216] The term "functional biological equivalent" of GHRH as used
herein is a polypeptide that has a distinct amino acid sequence
from a wild type GHRH polypeptide while simultaneously having
similar or improved biological activity when compared to the GHRH
polypeptide. The functional biological equivalent may be naturally
occurring or it may be modified by an individual. A skilled artisan
recognizes that the similar or improved biological activity as used
herein refers to facilitating and/or releasing growth hormone or
other pituitary hormones. A skilled artisan recognizes that in some
embodiments the encoded functional biological equivalent of GHRH is
a polypeptide that has been engineered to contain a distinct amino
acid sequence while simultaneously having similar or improved
biological activity when compared to the GHRH polypeptide. Methods
known in the art to engineer such a sequence include site-directed
mutagenesis.
[0217] The term "GeneSwitch.RTM. " (which is a registered trademark
of Valentis, Inc. (Burlingame, Calif.)) as used herein refers to
the technology of mifepristone-inducible heterologous nucleic acid
sequences encoding regulator proteins, GHRH, functional biological
equivalent or combination thereof. Such a technology is
schematically diagramed in FIG. 1A. A skilled artisan recognizes
that antiprogesterone agent alternatives to mifepristone are
available, including onapristone, ZK112993, ZK98734, and
5.quadrature.pregnane-3,2-dione.
[0218] The term "growth hormone" ("GH") as used herein is defined
as a hormone that relates to growth and acts as a chemical
messenger to exert its action on a target cell. In a specific
embodiment, the growth hormone is released by the action of growth
hormone releasing hormone.
[0219] The term "growth hormone releasing hormone" ("GHRH") as used
herein is defined as a hormone that facilitates or stimulates
release of growth hormone, and in a lesser extent other pituitary
hormones, such as prolactin. It is understood that the GHRH, the
recombinant GHRH, or a functional biological equivalent are
biologically active.
[0220] The term "heterologous nucleic acid sequence" as used herein
is defined as a DNA sequence comprising differing regulatory and
expression elements.
[0221] The term "lean body mass" ("LBM") as used herein is defined
as the mass of the body of an animal attributed to non-fat tissue
such as muscle.
[0222] The term "modified cells" as used herein is defined as the
cells from a subject that have an additional nucleic acid sequence
introduced into the cell.
[0223] The term "modified-donor-cells" as used herein refers to any
donor-cells that harbor a GHRH encoding nucleic acid sequence.
[0224] The term "molecular switch" as used herein refers to a
molecule that is delivered into a subject that can regulate
transcription of a gene. A skilled artisan recognizes that there
are many such switches known in the art, such as a tetracycline
switch, a zinc finger switch, a glucocorticoid switch, and so
forth.
[0225] The term "nucleic acid expression construct" as used herein
refers to any type of genetic construct comprising a nucleic acid
coding for a RNA capable of being transcribed. The term "expression
vector" can also be used interchangeably herein. In specific
embodiments, the nucleic acid expression construct comprises: a
promoter; a nucleotide sequence of interest; and a 3' untranslated
region; wherein the promoter, the nucleotide sequence of interest,
and the 3' untranslated region are operatively linked; and in vivo
expression of the nucleotide sequence of interest is regulated by
the promoter.
[0226] The term "operatively linked" as used herein refers to
elements or structures in a nucleic acid sequence that are linked
by operative ability and not physical location. The elements or
structures are capable of, or characterized by accomplishing a
desired operation. It is recognized by one of ordinary skill in the
art that it is not necessary for elements or structures in a
nucleic acid sequence to be in a tandem or adjacent order to be
operatively linked.
[0227] The term "poly-L-glutamate ("PLG")" as used herein refers to
a biodegradable polymer of L-glutamic acid that is suitable for use
as a vector or adjuvant for DNA transfer into cells with or without
electroporation.
[0228] The term "post-injection" as used herein refers to a time
period following the introduction of a nucleic acid cassette (that
contains heterologous nucleic acid sequence encoding GHRH or
functional biological equivalent thereof) into the cells of a
subject and the allowing of the expression of the encoded gene to
occur while the modified cells are within the living organism.
[0229] The term "plasmid" as used herein refers generally to a
construction comprised of extra-chromosomal genetic material,
usually of a circular duplex of DNA that can replicate
independently of chromosomal DNA. Plasmids, or fragments thereof,
may be used as vectors. Plasmids are double-stranded DNA molecule
that occur or are derived from bacteria and (rarely) other
microorganisms. However, mitochondrial and chloroplast DNA, yeast
killer and other cases are commonly excluded.
[0230] The term "plasmid mediated gene supplementation" as used
herein refers a method to allow a subject to have prolonged
exposure to a therapeutic range of a therapeutic protein by
utilizing a nucleic acid expression construct in vivo.
[0231] The term "pulse voltage device," or "pulse voltage injection
device" as used herein relates to an apparatus that is capable of
causing or causes uptake of nucleic acid molecules into the cells
of an organism by emitting a localized pulse of electricity to the
cells. The cell membrane then destabilizes, forming passageways or
pores. Conventional devices of this type are calibrated to allow
one to select or adjust the desired voltage amplitude and the
duration of the pulsed voltage. The primary importance of a pulse
voltage device is the capability of the device to facilitate
delivery of compositions of the invention, particularly linear DNA
fragments, into the cells of the organism.
[0232] The term "plasmid backbone" as used herein refers to a
sequence of DNA that typically contains a bacterial origin of
replication, and a bacterial antibiotic selection gene, which are
necessary for the specific growth of only the bacteria that are
transformed with the proper plasmid. However, there are plasmids,
called mini-circles, that lack both the antibiotic resistance gene
and the origin of replication (Darquet et al., 1997; Darquet et
al., 1999; Soubrier et al., 1999). The use of in vitro amplified
expression plasmid DNA (i.e. non-viral expression systems) avoids
the risks associated with viral vectors. The non-viral expression
systems products generally have low toxicity due to the use of
"species-specific" components for gene delivery, which minimizes
the risks of immunogenicity generally associated with viral
vectors. One aspect of the current invention is that the plasmid
backbone does not contain viral nucleotide sequences.
[0233] The term "promoter" as used herein refers to a sequence of
DNA that directs the transcription of a gene. A promoter may direct
the transcription of a prokaryotic or eukaryotic gene. A promoter
may be "inducible", initiating transcription in response to an
inducing agent or, in contrast, a promoter may be "constitutive",
whereby an inducing agent does not regulate the rate of
transcription. A promoter may be regulated in a tissue-specific or
tissue-preferred manner, such that it is only active in
transcribing the operable linked coding region in a specific tissue
type or types.
[0234] The term "rate of bone healing" as used herein is defined as
the time required to repair a bone fracture.
[0235] The term "recipient-subject" as used herein refers to any
species of the animal kingdom wherein modified-donor-cells can be
introduced from a donor-subject.
[0236] The term "regulator protein" as used herein refers to a
protein that increases or facilitates transcription of a target
nucleic acid sequence.
[0237] The term "residual linear plasmid backbone" as used herein
comprises any fragment of the plasmid backbone that is left at the
end of the process making the nucleic acid expression plasmid
linear.
[0238] The terms "subject" or "animal" as used herein refers to any
species of the animal kingdom. In preferred embodiments, it refers
more specifically to humans and domesticated animals used for: pets
(e.g. cats, dogs, etc.); work (e.g. horses, etc.); food (cows,
chicken, fish, lambs, pigs, etc); and all others known in the
art.
[0239] The term "tissue" as used herein refers to a collection of
similar cells and the intercellular substances surrounding them. A
skilled artisan recognizes that a tissue is an aggregation of
similarly specialized cells for the performance of a particular
function. For the scope of the present invention, the term tissue
does not refer to a cell line, a suspension of cells, or a culture
of cells. In a specific embodiment, the tissue is electroporated in
vivo. In another embodiment, the tissue is not a plant tissue. A
skilled artisan recognizes that there are four basic tissues in the
body: 1) epithelium; 2) connective tissues, including blood, bone,
and cartilage; 3) muscle tissue; and 4) nerve tissue. In a specific
embodiment, the methods and compositions are directed to transfer
of linear DNA into a muscle tissue by electroporation.
[0240] The term "therapeutic element" as used herein comprises
nucleic acid sequences that will lead to an in vivo expression of
an encoded gene product. One skilled in the art of molecular
biology will recognize that the therapeutic element may include,
but is not limited to a promoter sequence, a transgene, a poly A
sequence, or a 3' or 5' UTR.
[0241] The term "transfects" as used herein refers to introduction
of a nucleic acid into a eukaryotic cell. In some embodiments, the
cell is not a plant tissue or a yeast cell.
[0242] The term "viral backbone" as used herein refers to a nucleic
acid sequence that does not contain a promoter, a gene, and a 3'
poly A signal or an untranslated region, but contain elements
including, but not limited at site-specific genomic integration Rep
and inverted terminal repeats ("ITRs") or the binding site for the
tRNA primer for reverse transcription, or a nucleic acid sequence
component that induces a viral immunogenicity response when
inserted in vivo, allows integration, affects specificity and
activity of tissue specific promoters, causes transcriptional
silencing or poses safety risks to the subject.
[0243] The term "vascular pressure pulse" refers to a pulse of
pressure from a large volume of liquid to facilitate uptake of a
vector into a cell. A skilled artisan recognizes that the amount
and duration of the vascular pressure pulse is dependent on the
tissue, size, and overall health of the recipient animal, and
furthermore knows how to determine such parameters empirically.
[0244] The term "vector" as used herein refers to a construction
comprised of genetic material designed to direct transformation of
a targeted cell by delivering a nucleic acid sequence into that
cell. A vector may contain multiple genetic elements positionally
and sequentially oriented with other necessary elements such that
an included nucleic acid cassette can be transcribed and when
necessary translated in the transfected cells. These elements are
operatively linked. The term "expression vector" refers to a DNA
plasmid that contains all of the information necessary to produce a
recombinant protein in a heterologous cell.
[0245] The present invention concerns a method for decreasing the
body fat proportion, increasing lean body mass ("LBM"), increasing
bone density, increasing the rate of bone healing, or a combination
thereof, of an animal subject. In general the present invention can
be accomplished by delivering a nucleic acid sequence encoding GHRH
or functional biological equivalent thereof into the cells of the
subject (e.g. somatic, stem, or germ cells) and allowing expression
of the encoded gene to occur while the modified cells are within
the living organism. The subsequent expression of the GHRH or
functional biological equivalent thereof is regulated by a tissue
specific promoter (e.g. muscle), and/or by a regulator protein that
contains a modified ligand binding domain (e.g. molecular switch),
which will only be active when the correct modified ligand (e.g.
mifepistone) is administered to the subject. The expression and
ensuing release of GHRH or functional biological equivalent thereof
by the modified cells within the living organism will decrease the
body fat proportion, increase the LBM, increase the bone density,
and/or increase the bone healing rate of the subject.
[0246] One aspect of the current invention is a method for altering
lean body mass in a subject by utilizing a nucleic acid expression
vector regulated by a constitutive promoter. The method comprises
delivering into cells of the subject the nucleic acid expression
construct that encodes a growth-hormone-releasing-hormone ("GHRH")
or functional biological equivalent thereof. In a specific
embodiment, the nucleic acid expression construct is delivered into
the cells of the subject via electroporation, wherein the cells
comprise somatic, stem or germ cells. In another specific
embodiment the nucleic acid expression construct comprises SeqID
No: 11, SeqID No: 12, SeqID No: 13, SeqID No: 14, SeqID No: 17,
SeqID No: 18, SeqID No: 19, SeqID No: 20, or SeqID No: 21.
Transfection of the nucleic acid expression construct can be
expedited by utilizing a transfection-facilitating polypeptide
(e.g. charged polypeptide or poly-L-glutamate). The encoded GHRH or
functional biological equivalent thereof are expressed in tissue
specific cells of the subject, which comprises muscle cells. The
encoded GHRH or the encoded functional biological equivalent of
GHRH are biologically active polypeptides that have been engineered
to contain a distinct amino acid sequence while simultaneously
having similar or improved biologically activity when compared to
the GHRH polypeptide. In a preferred embodiment the encoded GHRH or
functional biological equivalent thereof is of formula (SEQID No:
6). Additionally, the encoded GHRH or functional biological
equivalent thereof facilitates growth hormone ("GH") secretion in
the subject.
[0247] A second aspect of the current invention is a method for
altering lean body mass in a subject by utilizing a nucleic acid
expression vector regulated by a molecular switch molecule. The
method comprises steps of delivering into cells of the subject a
first nucleic acid expression construct (SeqID No: 26), a second
nucleic acid expression construct (SeqID No: 27), and a molecular
switch; wherein the first nucleic acid expression construct encodes
growth-hormone-releasing-hormone ("GHRH") or functional biological
equivalent thereof; and wherein the second nucleic acid expression
construct has an encoding region of a regulator protein; and
delivering a molecular switch molecule into the subject, wherein
the molecular switch molecule governs activation of the regulator
protein and the regulator protein governs the activation of the
first nucleic acid expression construct. In some specific
embodiments, the nucleic acid expression construct further
comprises a transfection-facilitating polypeptide (e.g. a charged
polypeptide or poly-L-glutamate). The delivering step of the first
nucleic acid and the second nucleic acid expression construct into
the cells of the subject is via electroporation. A specific
embodiment of this method comprises that delivering the nucleic
acid expression construct into the cells of the subject initiates
expression of the encoded regulatory protein, but the regulatory
protein is inactive. However, upon delivering a molecular switch
(e.g. mifepristone) into the subject, the regulatory protein
becomes active, and the active regulatory protein initiates
expression of the GHRH or functional biological equivalent encoded
on the first nucleic acid sequence. The encoded GHRH is a
biologically active polypeptide; and the encoded functional
biological equivalent of GHRH is a polypeptide that has been
engineered to contain a distinct amino acid sequence while
simultaneously having similar or improved biologically activity
when compared to the GHRH polypeptide. The encoded GHRH or
functional biological equivalent thereof is of formula (SEQID No:
6). The encoded GHRH or functional biological equivalent thereof
facilitates growth hormone ("GH") secretion in the subject. In
another specific embodiment, the first nucleic acid expression
vector encodes a polypeptide of sequence SeqID No: 1, SeqID No: 2,
SeqID No: 3, or SeqID No: 4.
[0248] A third aspect of the current invention is a altering lean
body mass in a subject comprising the steps of: delivering into a
subject a recombinant growth-hormone-releasing-hormone ("GHRH") or
a biological functional equivalent thereof, wherein the recombinant
GHRH is a biologically active polypeptide. In specific embodiments,
the recombinant functional biological equivalent of GHRH is a
polypeptide that has been engineered to contain a distinct amino
acid sequence while simultaneously having similar or improved
biologically activity when compared to the GHRH polypeptide. In
another specific embodiment, the recombinant GHRH or functional
biological equivalent thereof is of formula (SEQID No: 6). The
recombinant GHRH or functional biological equivalent thereof
facilitates growth hormone ("GH") secretion in the subject.
[0249] A fourth aspect of the current invention is a method for
altering bone properties in a subject by utilizing a nucleic acid
expression vector regulated by a constitutive promoter. The method
comprises delivering into cells of the subject the nucleic acid
expression construct that encodes a
growth-hormone-releasing-hormone ("GHRH") or functional biological
equivalent thereof. In a specific embodiment, the nucleic acid
expression construct is delivered into the cells of the subject via
electroporation, wherein the cells comprise somatic, stem or germ
cells. In another specific embodiment the nucleic acid expression
construct comprises SeqID No: 11, SeqID No: 12, SeqID No: 13, SeqID
No: 14, SeqID No: 17, SeqID No: 18, SeqID No: 19, SeqID No: 20, or
SeqID No: 21. Transfection of the nucleic acid expression construct
can be expedited by utilizing a transfection-facilitating
polypeptide (e.g. charged polypeptide or poly-L-glutamate). The
encoded GHRH or functional biological equivalent thereof are
expressed in tissue specific cells of the subject, which comprises
muscle cells. The encoded GHRH or the encoded functional biological
equivalent of GHRH are biologically active polypeptides that have
been engineered to contain a distinct amino acid sequence while
simultaneously having similar or improved biologically activity
when compared to the GHRH polypeptide. In a preferred embodiment
the encoded GHRH or functional biological equivalent thereof is of
formula (SEQID No: 6). Additionally, the encoded GHRH or functional
biological equivalent thereof facilitates growth hormone ("GH")
secretion in the subject.
[0250] A fifth aspect of the current invention is a method for
altering bone properties in a subject by utilizing a nucleic acid
expression vector regulated by a molecular switch molecule. The
method comprises steps of delivering into cells of the subject a
first nucleic acid expression construct, a second nucleic acid
expression construct, and a molecular switch; wherein the first
nucleic acid expression construct encodes
growth-hormone-releasing-hormone ("GHRH") or functional biological
equivalent thereof; and wherein the second nucleic acid expression
construct has an encoding region of a regulator protein; and
delivering a molecular switch molecule into the subject, wherein
the molecular switch molecule governs activation of the regulator
protein and the regulator protein governs the activation of the
first nucleic acid expression construct. In some specific
embodiments, the nucleic acid expression construct further
comprises a transfection-facilitating polypeptide (e.g. a charged
polypeptide or poly-L-glutamate). The delivering step of the first
nucleic acid and the second nucleic acid expression construct into
the cells of the subject is via electroporation. A specific
embodiment of this method comprises that delivering the nucleic
acid expression construct into the cells of the subject initiates
expression of the encoded regulatory protein, but the regulatory
protein is inactive. However, upon delivering a molecular switch
(e.g. mifepristone) into the subject, the regulatory protein
becomes active, and the active regulatory protein initiates
expression of the GHRH or functional biological equivalent encoded
on the first nucleic acid sequence. The encoded GHRH is a
biologically active polypeptide; and the encoded functional
biological equivalent of GHRH is a polypeptide that has been
engineered to contain a distinct amino acid sequence while
simultaneously having similar or improved biologically activity
when compared to the GHRH polypeptide. The encoded GHRH or
functional biological equivalent thereof is of formula (SEQID No:
6). The encoded GHRH or functional biological equivalent thereof
facilitates growth hormone ("GH") secretion in the subject. In
another specific embodiment, the first nucleic acid expression
vector encodes a polypeptide of sequence SeqID No: 1, SeqID No: 2,
SeqID No: 3, or SeqID No: 4.
[0251] A sixth aspect of the current invention is a method for
altering bone properties in a subject comprising the steps of:
delivering into a subject a recombinant
growth-hormone-releasing-hormone ("GHRH") or a biological
functional equivalent thereof, wherein the recombinant GHRH is a
biologically active polypeptide. In specific embodiments, the
recombinant functional biological equivalent of GHRH is a
polypeptide that has been engineered to contain a distinct amino
acid sequence while simultaneously having similar or improved
biologically activity when compared to the GHRH polypeptide. In
another specific embodiment, the recombinant GHRH or functional
biological equivalent thereof is of formula (SEQID No: 6). The
recombinant GHRH or functional biological equivalent thereof
facilitates growth hormone ("GH") secretion in the subject.
[0252] The plasmid-mediated supplementation of GHRH approach
described herein offers advantages over the limitations of directly
injecting recombinant GHRH protein. Expression of nucleic acid
sequences encoding novel functional biological equivalents of GHRH
that are serum protease resistant can be directed by an expression
plasmid controlled by a synthetic muscle-specific promoter.
Expression of such GHRH or functional biological equivalent thereof
elicited high GH and IGF-I levels in pigs following delivery by
intramuscular injection and in vivo electroporation (Draghia-Akli
et al., 1999). The process of in vivo electroporation may involve
externally supplied electrodes, or in the case of needles,
internally supplied electrodes to aid in the inclusion of desired
nucleotide sequences into the cells of the subject within the
living organism. Although in vivo electroporation is the preferred
method of introducing the heterologous nucleic acid encoding system
into the cells of the subject, other methods exist and are known by
a person skilled in the art (e.g. electroporation, lipofectamine,
calcium phosphate, ex vivo transformation, direct injection, DEAE
dextran, sonication loading, receptor mediated transfection,
microprojectile bombardment, etc.). For example, it is also
possible to introduce the nucleic acid sequence that encodes the
GHRH or functional biological equivalent thereof directly into the
cells of the subject by first removing the cells from the body of
the subject or donor, maintaining the cells in culture, then
introducing the nucleic acid encoding system by a variety of
methods (e.g. electroporation, lipofectamine, calcium phosphate, ex
vivo transformation, direct injection, DEAE dextran, sonication
loading, receptor mediated transfection, microprojectile
bombardment, etc.), and finally reintroducing the modified cells
into the original subject or other host subject (the ex vivo
method). The GHRH sequence can be cloned into an adenovirus vector
or an adeno-associated vector and delivered by simple intramuscular
injection, or intravenous or intra-arterial injection. Plasmid DNA
carrying the GHRH sequence can be complexed with cationic lipids or
liposomes and delivered intramuscularly, intravenously or
subcutaneously.
[0253] Administration as used herein refers to the route of
introduction of a vector or carrier of DNA into the body.
Administration can be directly to a target tissue or by targeted
delivery to the target tissue after systemic administration. In
particular, the present invention can be used for supplementing
GHRH by administration of the vector, such as a plasmid, to the
body in order to establish controlled expression of the specific
nucleic acid sequence within tissues at certain useful levels.
[0254] The preferred means for administration of vector and use of
formulations for delivery are described above. The preferred
embodiment is by in vivo electroporation.
[0255] The route of administration of any selected vector construct
will depend on the particular use for the expression vectors. In
general, a specific formulation for each vector construct used will
focus on vector uptake with regard to the particular targeted
tissue, followed by demonstration of efficacy. Uptake studies will
include uptake assays to evaluate cellular uptake of the vectors
and expression of the tissue specific DNA of choice. Such assays
will also determine the localization of the target DNA after
uptake, and establishing the requirements for maintenance of
steady-state concentrations of expressed protein. Efficacy and
cytotoxicity can then be tested. Toxicity will not only include
cell viability but also cell function.
[0256] Muscle cells have the unique ability to take up DNA from the
extracellular space after simple injection of DNA particles as a
solution, suspension, or colloid into the muscle. Expression of DNA
by this method can be sustained for several months. DNA uptake in
muscle cells is further enhanced by utilizing in vivo
electroporation.
[0257] Delivery of formulated DNA vectors involves incorporating
DNA into macromolecular complexes that undergo endocytosis by the
target cell. Such complexes may include lipids, proteins,
carbohydrates, synthetic organic compounds, or inorganic compounds.
The characteristics of the complex formed with the vector (size,
charge, surface characteristics, composition) determines the
bioavailability of the vector within the body. Other elements of
the formulation function as ligand which interact with specific
receptors on the surface or interior of the cell. Other elements of
the formulation function to enhance entry into the cell, release
from the endosome, and entry into the nucleus.
[0258] Delivery can also be through use of DNA transporters. DNA
transporters refers to molecules which bind to DNA vectors and are
capable of being taken up by epidermal cells. DNA transporters
contain a molecular complex capable of non-covalently binding to
DNA and efficiently transporting the DNA through the cell membrane.
It is preferable that the transporter also transport the DNA
through the nuclear membrane. See, e.g., the following applications
all of which (including drawings) are hereby incorporated by
reference herein: (1) Woo et al., U.S. Pat. No. 6,150,168 entitled:
"A DNA Transporter System and Method of Use;" (2) Woo et al.,
PCT/US93/02725, entitled "A DNA Transporter System and method of
Use", filed Mar. 19, 1993; (3) Woo et al., U.S. Pat. No. 6,177,554
"Nucleic Acid Transporter Systems and Methods of Use;" (4) Szoka et
al., U.S. Pat. No. 5,955,365 entitled "Self-Assembling
Polynucleotide Delivery System;" and (5) Szoka et al.,
PCT/US93/03406, entitled "Self-Assembling Polynucleotide Delivery
System", filed Apr. 5, 1993.
[0259] Another method of delivery involves a DNA transporter
system. The DNA transporter system consists of particles containing
several elements that are independently and non-covalently bound to
DNA. Each element consists of a ligand which recognizes specific
receptors or other functional groups such as a protein complexed
with a cationic group that binds to DNA. Examples of cations which
may be used are spermine, spermine derivatives, histone, cationic
peptides and/or polylysine. One element is capable of binding both
to the DNA vector and to a cell surface receptor on the target
cell. Examples of such elements are organic compounds which
interact with the asialoglycoprotein receptor, the folate receptor,
the mannose-6-phosphate receptor, or the carnitine receptor. A
second element is capable of binding both to the DNA vector and to
a receptor on the nuclear membrane. The nuclear ligand is capable
of recognizing and transporting a transporter system through a
nuclear membrane. An example of such a ligand is the nuclear
targeting sequence from SV40 large T antigen or histone. A third
element is capable of binding to both the DNA vector and to
elements which induce episomal lysis. Examples include inactivated
virus particles such as adenovirus, peptides related to influenza
virus hemagglutinin, or the GALA peptide described in the Skoka
patent cited above.
[0260] Administration may also involve lipids. The lipids may form
liposomes which are hollow spherical vesicles composed of lipids
arranged in unilamellar, bilamellar, or multilamellar fashion and
an internal aqueous space for entrapping water soluble compounds,
such as DNA, ranging in size from 0.05 to several microns in
diameter. Lipids may be useful without forming liposomes. Specific
examples include the use of cationic lipids and complexes
containing DOPE which interact with DNA and with the membrane of
the target cell to facilitate entry of DNA into the cell.
[0261] Gene delivery can also be performed by transplanting
genetically engineered cells. For example, immature muscle cells
called myoblasts may be used to carry genes into the muscle fibers.
Myoblast genetically engineered to express recombinant human growth
hormone can secrete the growth hormone into the animal's blood.
Secretion of the incorporated gene can be sustained over periods up
to 3 months.
[0262] Myoblasts eventually differentiate and fuse to existing
muscle tissue. Because the cell is incorporated into an existing
structure, it is not only tolerated but nurtured. Myoblasts can
easily be obtained by taking muscle tissue from an individual who
needs supplementation of GHRH, and the genetically engineered cells
can also be easily put back with out causing damage to the patient
Is muscle. Similarly, keratinocytes may be used to delivery genes
to tissues. Large numbers of keratinocytes can be generated by
cultivation of a small biopsy. The cultures can be prepared as
stratified sheets and when grafted to humans, generate epidermis
which continues to improve in histotypic quality over many years.
The keratinocytes are genetically engineered while in culture by
transfecting the keratinocytes with the appropriate vector.
Although keratinocytes are separated from the circulation by the
basement membrane dividing the epidermis from the dermis, human
keratinocytes secrete into circulation the protein produced.
[0263] Delivery may also involve the use of viral vectors. For
example, an adenoviral vector may be constructed by replacing the
E1 region of the virus genome with the vector elements described in
this invention including promoter, 5'UTR, 3'UTR and nucleic acid
cassette and introducing this recombinant genome into 293 cells
which will package this gene into an infectious virus particle.
Virus from this cell may then be used to infect tissue ex vivo or
in vivo to introduce the vector into tissues leading to expression
of the gene in the nucleic acid cassette.
[0264] Although not wanting to be bound by theory, it is believed
that in order to provide an acceptable safety margin for the use of
such heterologous nucleic acid sequences in humans, a regulated
gene expression system is mandated to possess low levels of basal
expression of GHRH, and still retain a high inducibility. Thus,
target gene expression can be regulated by incorporating molecular
switch technology as schematically diagramed in FIG. 1A. The
commercially available GeneSwitch.RTM. system for ligand-dependent
induction of transgene expression is based on a C-terminally
truncated progesterone receptor that fails to bind to its natural
agonist, progesterone, but instead is activated by antiprogestins,
such as mifepristone ("MFP") (Vegeto et al., 1992; Xu et al.,
1996). Thus, the heterologous nucleic acid sequence introduced into
the cells of the subject requires MFP to be transcriptionally
activated. The chimeric regulator protein of the GeneSwitch.RTM.
system consists of the ligand binding domain of the truncated human
progesterone receptor that has been fused to the DNA binding domain
of the yeast GAL4 protein (which binds a specific 17 bp recognition
sequence) and a transcriptional activation domain from the p65
subunit of human NF-kB (Abruzzese et al., 1999; Abruzzese et al.,
2000). The gene for the GeneSwitch.RTM. regulator protein was
inserted into a myogenic expression vector, designated pGS1633,
which is expressed constitutively under the control of a
muscle-specific skeletal alpha-actin ("SK") promoter The GHRH
plasmid, designated, p6.times.Gal4/TATA-GHRH, or pGHRH1633 contains
an inducible promoter that consists of six copies of the 17-mer
Gal4 binding site fused to a minimal TATA box promoter. The GHRH
coding sequence is a 228-bp fragment of super-porcine mutated GHRH
cDNA, termed HV-GHRH (Draghia-Akli et al., 1999). The HV-GHRH
molecule displays a high degree of stability in serum, with a
half-life of 6 hours, versus the natural GHRH, that has a 6-12 min
half-life. The muscle-specific GeneSwitch.RTM. and inducible GHRH
plasmids both have a 5' untranslated region that contains a
synthetic intron, and a 3' untranslated region/poly(A) site that is
from the human GH gene.
[0265] Recombinant GH replacement therapy is widely used
clinically, with beneficial effects, but generally, the doses are
supraphysiological. Such elevated doses of recombinant GH are
associated with deleterious side-effects, for example, up to 30% of
the recombinant GH treated patients report a higher frequency of
insulin resistance (Blethen, 1995; Verhelst et al., 1997) or
accelerated bone epiphysis growth and closure in pediatric patients
(Blethen and Rundle, 1996). In addition, molecular heterogeneity of
circulating GH may have important implications in growth and
homeostasis, which can lead to a less potent GH that has a reduced
ability to stimulate the prolactin receptor; it has also been
described that the 20 kDa form of GH has less potency to cause
urine retention than the 22 kDa form (Satozawa et al., 2000;
Tsunekawa et al., 1999; Wada et al., 1998). These unwanted side
effects result from the fact that treatment with recombinant
exogenous GH protein raises basal levels of GH and abolishes the
natural episodic pulses of GH. In contradistinction, no side
effects have been reported for recombinant GHRH therapies. The
normal levels of GHRH in the pituitary portal circulation range
from about 150-to-800 pg/ml, while systemic circulating values of
the hormone are up to about 100-500 pg/ml. Some patients with
acromegaly caused by extracranial tumors have level that is nearly
10 times as high (e.g. 50 ng/ml of immunoreactive GHRH) (Thorner et
al., 1984). Long-term studies using recombinant GHRH therapies (1-5
years) in children and elderly humans have shown an absence of the
classical GH side-effects, such as changes in fasting glucose
concentration or, in pediatric patients, the accelerated bone
epiphysal growth and closure or slipping of the capital femoral
epiphysis (Chevalier et al., 2000) (Duck et al., 1992; Vittone et
al., 1997). Numerous studies in humans, sheep or pigs showed that
continuous infusion with recombinant GHRH protein restores the
normal GH pattern without desensitizing GHRH receptors or depleting
GH supplies (Dubreuil et al., 1990b). As this system is capable of
a degree of feed-back which is abolished in the GH therapies, GHRH
recombinant protein therapy may be more physiological than GH
therapy. However, due to the short half-life of GHRH in vivo,
frequent (one to three times per day) intravenous, subcutaneous or
intranasal (requiring 300-fold higher dose) administrations are
necessary (Evans et al., 1985; Thorner et al., 1986). Thus, as a
chronic therapy, recombinant GHRH protein administration is not
practical. A gene transfer approach, however could overcome this
limitations to GHRH use. Moreover, a wide range of doses can be
therapeutic. The choice of GHRH for a gene therapeutic application
is favored by the fact that the gene, cDNA and native and several
mutated molecules have been characterized for the pig and other
species (Bohlen et al., 1983; Guillemin et al., 1982), and the
measurement of therapeutic efficacy is straightforward and
unequivocal.
[0266] Among the non-viral techniques for gene transfer in vivo,
the direct injection of plasmid DNA into muscle is simple,
inexpensive, and safe. The inefficient DNA uptake into muscle
fibers after simple direct injection hag led to relatively low
expression levels (Prentice et al., 1994; Wells et al., 1997) In
addition, the duration of the transgene expression has been short
(Wolff et al., 1990). The most successful previous clinical
applications have been confined to vaccines (Danko and Wolff, 1994;
Tsurumi et al., 1996). Recently, significant progress to enhance
plasmid delivery in vivo and subsequently to achieve physiological
levels of a secreted protein was obtained using the electroporation
technique. Recently, significant progress has been obtained using
electroporation to enhance plasmid delivery in vivo.
Electroporation has been used very successfully to transfect tumor
cells after injection of plasmid (Lucas et al., 2002; Matsubara et
al., 2001) or to deliver the anti-tumor drug bleomycin to cutaneous
and subcutaneous tumors in humans (Gehl et al., 1998; Heller et
al., 1996). Electroporation also has been extensively used in mice
(Lesbordes et al., 2002; Lucas et al., 2001; Vilquin et al., 2001),
rats (Terada et al., 2001; Yasui et al., 2001), and dogs (Fewell et
al., 2001) to deliver therapeutic genes that encode for a variety
of hormones, cytokines or enzymes. Our previous studies using
growth hormone releasing hormone (GHRH) showed that plasmid therapy
with electroporation is scalable and represents a promising
approach to induce production and regulated secretion of proteins
in large animals and humans (Draghia-Akli et al., 1999;
Draghia-Akli et al., 2002). Electroporation also has been
extensively used in rodents and other small animals (Bettan et al.,
2000; Yin and Tang, 2001). It has been observed that the electrode
configuration affects the electric field distribution, and
subsequent results (Gehl et al., 1999; Miklavcic et al., 1998).
Preliminary experiments indicated that for a large animal model,
needle electrodes give consistently better reproducible results
than external caliper electrodes.
[0267] Combining the powerful electroporation delivery method with
an improved plasmid DNA vector system produced significant changes
that decreased the body fat proportion, increased lean body mass
("LBM"), or both, in an animal, such as a large animal, at very low
plasmid dosage.
I. Vectors
[0268] 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 wherein, in some embodiments, it can be
replicated. A nucleic acid sequence can be native to the animal, or
it 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), linear DNA fragments, and
artificial chromosomes (e.g., YACs), although in a preferred
embodiment the vector contains substantially no viral sequences.
One of skill in the art would be well equipped to construct a
vector through standard recombinant techniques (see, for example,
(Sambrook et al., 1989).
[0269] The term "expression vector" refers to any type of genetic
construct comprising a nucleic acid coding for a RNA capable of
being transcribed. 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 operatively linked coding sequence in a particular host cell.
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.
II. Plasmid Vectors
[0270] In certain embodiments, a linear DNA fragment from a plasmid
vector is contemplated for use to transfect a eukaryotic cell,
particularly a mammalian cell. In general, plasmid vectors
containing replicon and control sequences which are derived from
species compatible with the host cell are used in connection with
these hosts. The vector ordinarily carries a replication site, as
well as marking sequences which are capable of providing phenotypic
selection in transformed cells. In a non-limiting example, E. coli
is often transformed using derivatives of pBR322, a plasmid derived
from an E. coli species. pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR plasmid, or other microbial
plasmid or phage must also contain, or be modified to contain, for
example, promoters which can be used by the microbial organism for
expression of its own proteins. A skilled artisan recognizes that
any plasmid in the art may be modified for use in the methods of
the present invention. In a specific embodiment, for example, a
GHRH vector used for the therapeutic applications is derived from
pBlueScript KS+ and has a kanamycin resistance gene.
[0271] In addition, phage vectors containing replicon and control
sequences that are compatible with the host microorganism can be
used as transforming vectors in connection with these hosts. For
example, the phage lambda GEM.TM.-11 may be utilized in making a
recombinant phage vector which can be used to transform host cells,
such as, for example, E. coli LE392.
[0272] Further useful plasmid vectors include pIN vectors (Inouye
et al., 1985); and pGEX vectors, for use in generating glutathione
S-transferase ("GST") soluble fusion proteins for later
purification and separation or cleavage. Other suitable fusion
proteins are those with .beta.-galactosidase, ubiquitin, and the
like.
[0273] Bacterial host cells, for example, E. coli, comprising the
expression vector, are grown in any of a number of suitable media,
for example, LB. The expression of the recombinant protein in
certain vectors may be induced, as would be understood by those of
skill in the art, by contacting a host cell with an agent specific
for certain promoters, e.g., by adding IPTG to the media or by
switching incubation to a higher temperature. After culturing the
bacteria for a further period, generally of between 2 and 24 h, the
cells are collected by centrifugation and washed to remove residual
media.
III. Promoters and Enhancers
[0274] A promoter is a control sequence that is a region of a
nucleic acid sequence at which initiation and rate of transcription
of a gene product are controlled. It may contain genetic elements
at which regulatory proteins and molecules may bind, such as RNA
polymerase and other transcription factors, to initiate the
specific transcription a nucleic acid sequence. 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.
[0275] A promoter generally comprises a sequence that functions to
position the start site for RNA synthesis. The best known example
of this is the TATA box, but in some promoters lacking a TATA box,
such as, for example, the promoter for the mammalian terminal
deoxynucleotidyl transferase gene and the promoter for the SV40
late genes, a discrete element overlying the start site itself
helps to fix the place of initiation. Additional promoter elements
regulate the frequency of transcriptional initiation. Typically,
these are located in the region 30-110 bp upstream of the start
site, although a number of promoters have been shown to contain
functional elements downstream of the start site as well. To bring
a coding sequence "under the control of" a promoter, one positions
the 5' end of the transcription initiation site of the
transcriptional reading frame "downstream" of (i.e., 3' of) the
chosen promoter. The "upstream" promoter stimulates transcription
of the DNA and promotes expression of the encoded RNA.
[0276] The spacing between promoter elements frequently is
flexible, so that promoter function is preserved when elements are
inverted or moved relative to one another. In the tk promoter, the
spacing between promoter elements can be increased to 50 bp apart
before activity begins to decline. Depending on the promoter, it
appears that individual elements can function either cooperatively
or independently to activate transcription. 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.
[0277] A promoter may be one naturally associated with a nucleic
acid 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,
synthetic or heterologous promoter, which refers to a promoter that
is not normally associated with a nucleic acid sequence in its
natural environment. A recombinant, synthetic 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 virus, or
prokaryotic 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. For example, promoters that are most commonly
used in recombinant DNA construction include the .beta.-lactamase
(penicillinase), lactose and tryptophan (trp) promoter systems. 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. Nos. 4,683,202 and 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.
[0278] Naturally, it will be important to employ a promoter and/or
enhancer that effectively directs the expression of the DNA segment
in the organelle, cell type, tissue, organ, or 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, (see, for example (Sambrook et
al., 1989)). 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.
[0279] Additionally any promoter/enhancer combination (as per, for
example, the Eukaryotic Promoter Data Base EPDB,
http://www.epd.isb-sib.ch/) could also be used to drive expression.
Use of a T3, T7 or SP6 cytoplasmic expression system is another
possible embodiment. Eukaryotic cells can support cytoplasmic
transcription from certain bacterial promoters if the appropriate
bacterial polymerase is provided, either as part of the delivery
complex or as an additional genetic expression construct.
[0280] Tables 1 and 2 list non-limiting examples of
elements/promoters that may be employed, in the context of the
present invention, to regulate the expression of a RNA. Table 2
provides non-limiting examples of inducible elements, which are
regions of a nucleic acid sequence that can be activated in
response to a specific stimulus.
TABLE-US-00001 TABLE 1 Promoter and/or Enhancer Promoter/Enhancer
Relevant References Immunoglobulin Heavy Chain Immunoglobulin Light
Chain T-Cell Receptor HLA DQ a and/or DQ .beta. .beta.-Interferon
Interleukin-2 Interleukin-2 Receptor MHC Class II 5 MHC Class II
HLA-Dra .beta.-Actin (Kawamoto et al., 1988; Kawamoto et al., 1989)
Muscle Creatine Kinase (MCK) (Horlick and Benfield, 1989; Jaynes et
al., 1988) Prealbumin (Transthyretin) Elastase I Metallothionein
(MTII) (Inouye et al., 1994; Narum et al., 2001; Skroch et al.,
1993) Collagenase Albumin (Pinkert et al., 1987; Tronche et al.,
1989) .alpha.-Fetoprotein .gamma.-Globin .beta.-Globin (Tronche et
al., 1990; Trudel and Costantini, 1987) c-fos c-HA-ras Insulin
(German et al., 1995; Ohlsson et al., 1991) Neural Cell Adhesion
Molecule (NCAM) .alpha..sub.1-Antitrypsin H2B (TH2B) Histone Mouse
and/or Type I Collagen Glucose-Regulated Proteins (GRP94 and GRP78)
Rat Growth Hormone (Larsen et al., 1986) Human Serum Amyloid A
(SAA) Troponin I (TN I) (Lin et al., 1991; Yutzey and Konieczny,
1992) Platelet-Derived Growth Factor (Pech et al., 1989) (PDGF)
Duchenne Muscular Dystrophy (Klamut et al., 1990; Klamut et al.,
1996) SV40 Polyoma Retroviruses Papilloma Virus Hepatitis B Virus
Human Immunodeficiency Virus Cytomegalovirus (CMV) (Boshart et al.,
1985; Dorsch-Hasler et al., 1985) Gibbon Ape Leukemia Virus
Synthetic muscle specific promoters (Draghia-Akli et al., 1999;
Draghia-Akli et al., 2002; Li (c5-12, c1-28) et al., 1999)
TABLE-US-00002 TABLE 2 Element/Inducer Element Inducer MT II
Phorbol Ester (TFA) Heavy metals MMTV (mouse mammary tumor virus)
Glucocorticoids .beta.-Interferon Poly(rI)x/Poly(rc) Adenovirus 5
E2 ElA Collagenase Phorbol Ester (TPA) Stromelysin Phorbol Ester
(TPA) SV40 Phorbol Ester (TPA) Murine MX Gene Interferon, Newcastle
Disease Virus GRP78 Gene A23187 .alpha.-2-Macroglobulin IL-6
Vimentin Serum MHC Class I Gene H-2.kappa.b Interferon HSP70 ElA,
SV40 Large T Antigen Proliferin Phorbol Ester-TPA Tumor Necrosis
Factor .alpha. PMA Thyroid Stimulating Hormone .alpha. Gene Thyroid
Hormone
[0281] 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. Nonlimiting 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 (Liu et
al., 2000; Tsumaki et al., 1998), D1A dopamine receptor gene (Lee
et al., 1997), insulin-like growth factor II (Dai et al., 2001; Wu
et al., 1997), and human platelet endothelial cell adhesion
molecule-1 (Almendro et al., 1996).
[0282] In a preferred embodiment, a synthetic muscle promoter is
utilized, such as SPc5-12 (Li et al., 1999), which contains a
proximal serum response element ("SRE") from skeletal
.quadrature.-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). In a preferred embodiment,
the promoter utilized in the invention does not get shut off or
reduced in activity significantly by endogenous cellular machinery
or factors. Other elements, including trans-acting factor binding
sites and enhancers may be used in accordance with this embodiment
of the invention. In an alternative embodiment, a natural myogenic
promoter is utilized, and a skilled artisan is aware how to obtain
such promoter sequences from databases including the National
Center for Biotechnology Information ("NCBI") GenBank database or
the NCBI PubMed site. A skilled artisan is aware that these
databases may be utilized to obtain sequences or relevant
literature related to the present invention.
IV. Initiation Signals and Internal Ribosome Binding Sites
[0283] 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.
[0284] 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.quadrature. 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, each herein incorporated by
reference).
V. Multiple Cloning Sites
[0285] Vectors can include a 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, for example, (Carbonelli et al., 1999;
Cocea, 1997; Levenson et al., 1998) 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.
VI. Splicing Sites
[0286] 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, for example, (Chandler et
al., 1997), herein incorporated by reference.) VII. Termination
Signals
[0287] The vectors or constructs of the present invention will
generally comprise at least one termination signal. A "termination
signal" or "terminator" is comprised of the DNA sequences involved
in specific termination of an RNA transcript by an RNA polymerase.
Thus, in certain embodiments a termination signal that ends the
production of an RNA transcript is contemplated. A terminator may
be necessary in vivo to achieve desirable message levels.
[0288] In eukaryotic systems, the terminator region may also
comprise specific DNA sequences that permit site-specific cleavage
of the new transcript so as to expose a polyadenylation site. This
signals a specialized endogenous polymerase to add a stretch of
about 200 A residues ("polyA") to the 3' end of the transcript. RNA
molecules modified with this polyA tail appear to more stable and
are translated more efficiently. Thus, in other embodiments
involving eukaryotes, it is preferred that that terminator
comprises a signal for the cleavage of the RNA, and it is more
preferred that the terminator signal promotes polyadenylation of
the message. The terminator and/or polyadenylation site elements
can serve to enhance message levels and to minimize read through
from the cassette into other sequences.
[0289] Terminators contemplated for use in the invention include
any known terminator of transcription described herein or known to
one of ordinary skill in the art, including but not limited to, for
example, the termination sequences of genes, such as for example
the bovine growth hormone terminator or viral termination
sequences, such as for example the SV40 terminator. In certain
embodiments, the termination signal may be a lack of transcribable
or translatable sequence, such as due to a sequence truncation.
VIII. Polyadenylation Signals
[0290] In expression, particularly eukaryotic 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 any such sequence may be
employed. Preferred embodiments include the SV40 polyadenylation
signal, skeletal alpha actin 3'UTR or the human or bovine growth
hormone polyadenylation signal, convenient and known to function
well in various target cells. Polyadenylation may increase the
stability of the transcript or may facilitate cytoplasmic
transport.
IX. Origins of Replication
[0291] 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.
X. Selectable and Screenable Markers
[0292] In certain embodiments of the invention, cells containing a
nucleic acid construct of the present invention 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.
[0293] 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 calorimetric 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.
XI. Electroporation
[0294] In certain embodiments of the present invention, a nucleic
acid is introduced into an organelle, a cell, a tissue or an
organism via electroporation. Electroporation involves the exposure
of a suspension of cells and DNA to a high-voltage electric
discharge. In some variants of this method, certain cell
wall-degrading enzymes, such as pectin-degrading enzymes, are
employed to render the target recipient cells more susceptible to
transformation by electroporation than untreated cells (U.S. Pat.
No. 5,384,253, incorporated herein by reference). Alternatively,
recipient cells can be made more susceptible to transformation by
mechanical wounding and other methods known in the art.
[0295] Transfection of eukaryotic cells using electroporation has
been quite successful. Mouse pre-B lymphocytes have been
transfected with human kappa-immunoglobulin genes (Potter et al.,
1984), and rat hepatocytes have been transfected with the
chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in
this manner.
[0296] The underlying phenomenon of electroporation is believed to
be the same in all cases, but the exact mechanism responsible for
the observed effects has not been elucidated. Although not wanting
to be bound by theory, the overt manifestation of the
electroporative effect is that cell membranes become transiently
permeable to large molecules, after the cells have been exposed to
electric pulses. There are conduits through cell walls, which under
normal circumstances, maintain a resting transmembrane potential of
ca. 90 mV by allowing bi-directional ionic migration.
[0297] Although not wanting to be bound by theory, electroporation
makes use of the same structures, by forcing a high ionic flux
through these structures and opening or enlarging the conduits. In
prior art, metallic electrodes are placed in contact with tissues
and predetermined voltages, proportional to the distance between
the electrodes are imposed on them. The protocols used for
electroporation are defined in terms of the resulting field
intensities, according to the formula E=V/d, where ("E") is the
field, ("V") is the imposed voltage and ("d") is the distance
between the electrodes.
[0298] The electric field intensity E has been a very important
value in prior art when formulating electroporation protocols for
the delivery of a drug or macromolecule into the cell of the
subject. Accordingly, it is possible to calculate any electric
field intensity for a variety of protocols by applying a pulse of
predetermined voltage that is proportional to the distance between
electrodes. However, a caveat is that an electric field can be
generated in a tissue with insulated electrodes (i.e. flow of ions
is not necessary to create an electric field). Although not wanting
to be bound by theory, it is the current that is necessary for
successful electroporation not electric field per se.
[0299] During electroporation, the heat produced is the product of
the interelectrode impedance, the square of the current, and the
pulse duration. Heat is produced during electroporation in tissues
and can be derived as the product of the inter-electrode current,
voltage and pulse duration. The protocols currently described for
electroporation are defined in terms of the resulting field
intensities E, which are dependent on short voltage pulses of
unknown current. Accordingly, the resistance or heat generated in a
tissue cannot be determined, which leads to varied success with
different pulsed voltage electroporation protocols with
predetermined voltages. The ability to limit heating of cells
across electrodes can increase the effectiveness of any given
electroporation voltage pulsing protocol. For example, prior art
teaches the utilization of an array of six needle electrodes
utilizing a predetermined voltage pulse across opposing electrode
pairs. This situation sets up a centralized pattern during an
electroporation event in an area where congruent and intersecting
overlap points develop. Excessive heating of cells and tissue along
electroporation path will kill the cells, and limit the
effectiveness of the protocol. However, symmetrically arranged
needle electrodes without opposing pairs can produce a
decentralized pattern during an electroporation event in an area
where no congruent electroporation overlap points can develop.
[0300] Controlling the current flow between electrodes allows one
to determine the relative heating of cells. Thus, it is the current
that determines the subsequent effectiveness of any given pulsing
protocol, and not the voltage across the electrodes. Predetermined
voltages do not produce predetermined currents, and prior art does
not provide a means to determine the exact dosage of current, which
limits the usefulness of the technique. Thus, controlling an
maintaining the current in the tissue between two electrodes under
a threshold will allow one to vary the pulse conditions, reduce
cell heating, create less cell death, and incorporate
macromolecules into cells more efficiently when compared to
predetermined voltage pulses.
[0301] One embodiment of the present invention to overcome the
above problem by providing a means to effectively control the
dosage of electricity delivered to the cells in the inter-electrode
space by precisely controlling the ionic flux that impinges on the
conduits in the cell membranes. The precise dosage of electricity
to tissues can be calculated as the product of the current level,
the pulse length and the number of pulses delivered. Thus, a
specific embodiment of the present invention can deliver the
electroporative current to a volume of tissue along a plurality of
paths without, causing excessive concentration of cumulative
current in any one location, thereby avoiding cell death owing to
overheating of the tissue.
[0302] Although not wanting to be bound by theory, the nature of
the voltage pulse to be generated is determined by the nature of
tissue, the size of the selected tissue and distance between
electrodes. It is desirable that the voltage pulse be as homogenous
as possible and of the correct amplitude. Excessive field strength
results in the lysing of cells, whereas a low field strength
results in reduced efficacy of electroporation. Some
electroporation devices utilize the distance between electrodes to
calculate the electric field strength and predetermined voltage
pulses for electroporation. This reliance on knowing the distance
between electrodes is a limitation to the design of electrodes.
Because the programmable current pulse controller will determine
the impedance in a volume of tissue between two electrodes, the
distance between electrodes is not a critical factor for
determining the appropriate electrical current pulse. Therefore, an
alternative embodiment of a needle electrode array design would be
one that is non-symmetrical. In addition, one skilled in the art
can imagine any number of suitable symmetrical and non-symmetrical
needle electrode arrays that do not deviate from the spirit and
scope of the invention. The depth of each individual electrode
within an array and in the desired tissue could be varied with
comparable results. In addition, multiple injection sites for the
macromolecules could be added to the needle electrode array.
XII. Restriction Enzymes
[0303] In some embodiments of the present invention, a linear DNA
fragment is generated by restriction enzyme digestion of a parent
DNA molecule. Examples of restriction enzymes are provided
below.
TABLE-US-00003 Name Recognition Sequence AatII GACGTC Acc65 I
GGTACC Acc I GTMKAC Aci I CCGC Acl I AACGTT Afe I AGCGCT Afl II
CTTAAG Afl III ACRYGT Age I ACCGGT Ahd I GACNNNNNGTC Alu I AGCT Alw
I GGATC AlwN I CAGNNNCTG Apa I GGGCCC ApaL I GTGCAC Apo I RAATTY
Asc I GGCGCGCC Ase I ATTAAT Ava I CYCGRG Ava II GGWCC Avr II CCTAGG
Bae I NACNNNGTAPyCN BamH I GGATCC Ban I GGYRCC Ban II GRGCYC Bbs I
GAAGAC Bbv I GCAGC BbvC I CCTCAGC Bcg I CGANNNNNNTGC BciV I GTATCC
Bcl I TGATCA Bfa I CTAG Bgl I GCCNNNNNGGC Bgl II AGATCT Blp I
GCTNAGC Bmr I ACTGGG Bpm I CTGGAG BsaA I YACGTR BsaB I GANNNNATC
BsaH I GRCGYC Bsa I GGTCTC BsaJ I CCNNGG BsaW I WCCGGW BseR I
GAGGAG Bsg I GTGCAG BsiE I CGRYCG BsiHKA I GWGCWC BsiW I CGTACG Bsl
I CCNNNNNNNGG BsmA I GTCTC BsmB I CGTCTC BsmF I GGGAC Bsm I GAATGC
BsoB I CYCGRG Bsp1286 I GDGCHC BspD I ATCGAT BspE I TCCGGA BspH I
TCATGA BspM I ACCTGC BsrB I CCGCTC BsrD I GCAATG BsrF I RCCGGY BsrG
I TGTACA Bsr I ACTGG BssH II GCGCGC BssK I CCNGG Bst4C I ACNGT BssS
I CACGAG BstAP I GCANNNNNTGC BstB I TTCGAA BstE II GGTNACC BstF5 I
GGATGNN BstN I CCWGG BstU I CGCG BstX I CCANNNNNNTGG BstY I RGATCY
BstZ17 I GTATAC Bsu36 I CCTNAGG Btg I CCPuPyGG Btr I CACGTG Cac8 I
GCNNGC Cla I ATCGAT Dde I CTNAG Dpn I GATC Dpn II GATC Dra I TTTAAA
Dra III CACNNNGTG Drd I GACNNNNNNGTC Eae I YGGCCR Eag I CGGCCG Ear
I CTCTTC Eci I GGCGGA EcoN I CCTNNNNNAGG EcoO109 I RGGNCCY EcoR I
GAATTC EcoR V GATATC Fau I CCCGCNNNN Fnu4H I GCNGC Fok I GGATG Fse
I GGCCGGCC Fsp I TGCGCA Hae II RGCGCY Hae III GGCC Hga I GACGC Hha
I GCGC Hinc II GTYRAC Hind III AAGCTT Hinf I GANTC HinP1 I GCGC Hpa
I GTTAAC Hpa II CCGG Hph I GGTGA Kas I GGCGCC Kpn I GGTACC Mbo I
GATC Mbo II GAAGA Mfe I CAATTG Mlu I ACGCGT Mly I GAGTCNNNNN Mnl I
CCTC Msc I TGGCCA Mse I TTAA Msl I CAYNNNNRTG
MspA1 I CMGCKG Msp I CCGG Mwo I GCNNNNNNNGC Nae I GCCGGC Nar I
GGCGCC Nci I CCSGG Nco I CCATGG Nde I CATATG NgoMI V GCCGGC Nhe I
GCTAGC Nla III CATG Nla IV GGNNCC Not I GCGGCCGC Nru I TCGCGA Nsi I
ATGCAT Nsp I RCATGY Pac I TTAATTAA PaeR7 I CTCGAG Pci I ACATGT PflF
I GACNNNGTC PflM I CCANNNNNTGG Ple I GAGTC Pme I GTTTAAAC Pml I
CACGTG PpuM I RGGWCCY PshA I GACNNNNGTC Psi I TTATAA PspG I CCWGG
PspOM I GGGCCC Pst I CTGCAG Pvu I CGATCG Pvu II CAGCTG Rsa I GTAC
Rsr II CGGWCCG Sac I GAGCTC Sac II CCGCGG Sal I GTCGAC Sap I
GCTCTTC Sau3A I GATC Sau96 I GGNCC Sbf I CCTGCAGG Sca I AGTACT ScrF
I CCNGG SexA I ACCWGGT SfaN I GCATC Sfc I CTRYAG Sfi I
GGCCNNNNNGGCC Sfo I GGCGCC SgrA I CRCCGGYG Sma I CCCGGG Sml I
CTYRAG SnaB I TACGTA Spe I ACTAGT Sph I GCATGC Ssp I AATATT Stu I
AGGCCT Sty I CCWWGG Swa I ATTTAAAT Taq I TCGA Tfi I GAWTC Tli I
CTCGAG Tse I GCWGC Tsp45 I GTSAC Tsp509 I AATT TspR I CAGTG Tthl11
I GACNNNGTC Xba I TCTAGA Xcm I CCANNNNNNNNNTGG Xho I CTCGAG Xma I
CCCGGG Xmn I GAANNNNTTC
[0304] The term "restriction enzyme digestion" of DNA as used
herein refers to catalytic cleavage of the DNA with an enzyme that
acts only at certain locations in the DNA. Such enzymes are called
restriction endonucleases, and the sites for which each is specific
is called a restriction site. The various restriction enzymes used
herein are commercially available and their reaction conditions,
cofactors, and other requirements as established by the enzyme
suppliers are used. Restriction enzymes commonly are designated by
abbreviations composed of a capital letter followed by other
letters representing the microorganism from which each restriction
enzyme originally was obtained and then a number designating the
particular enzyme. In general, about 1 .quadrature.g of plasmid or
DNA fragment is used with about 1-2 units of enzyme in about 20
.quadrature.l of buffer solution. Appropriate buffers and substrate
amounts for particular restriction enzymes are specified by the
manufacturer. Incubation of about 1 hour at 37.degree. C. is
ordinarily used, but may vary in accordance with the supplier's
instructions. After incubation, protein or polypeptide is removed
by extraction with phenol and chloroform, and the digested nucleic
acid is recovered from the aqueous fraction by precipitation with
ethanol. Digestion with a restriction enzyme may be followed with
bacterial alkaline phosphatase hydrolysis of the terminal 5'
phosphates to prevent the two restriction cleaved ends of a DNA
fragment from "circularizing" or forming a closed loop that would
impede insertion of another DNA fragment at the restriction site.
Unless otherwise stated, digestion of plasmids is not followed by
5' terminal dephosphorylation. Procedures and reagents for
dephosphorylation are conventional as described in the art.
EXAMPLES
[0305] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Construction of DNA Vectors and Methods in Animal Subject
[0306] In order to increase lean body mass, decrease body fat
proportions, increase bone density, and improve bone healing rate,
it was first necessary to design several GHRH expression
constructs. Briefly, the plasmid vectors contained the muscle
specific synthetic promoter SPc5-12 (Li et al., 1999) attached to a
wild type or analog porcine GHRH. The analog GHRH sequences were
generated by site directed mutagenesis as described in methods
section. Nucleic acid sequences encoding GHRH or analog were cloned
into the BamHI/HindIII sites of pSPc5-12 plasmid, to generate
pSP-GHRH. Other elements contained in the plasmids include a 3'
untranslated region ("3'UTR") (SEQ ID No: 8) of growth hormone and
an SV40 3'UTR from pSEAP-2 Basic Vector as described in the methods
section. The unique nucleic acid sequences for the constructs used
are shown in FIG. 1.
[0307] DNA constructs: Plasmid vectors containing the muscle
specific synthetic promoter SPc5-12 (SeqID No: 7) were previously
described (Li et al., 1999). Wild type and mutated porcine GHRH
cDNAs were generated by site directed mutagenesis of GHRH cDNA
(SeqID No: 9) (Altered Sites II in vitro Mutagenesis System,
Promega, Madison, Wis.), and cloned into the BamHI/Hind III sites
of pSPc5-12, to generate pSP-wt-GHRH (SeqID No: 15), or pSP-HV-GHRH
(SeqID No: 11), respectively. The 3' untranslated region (3 'UTR)
of growth hormone was cloned downstream of GHRH cDNA. The resultant
plasmids contained mutated coding region for GHRH, and the
resultant amino acid sequences were not naturally present in
mammals. Although not wanting to be bound by theory, the effects on
increased bone density, and increased healing rate of bone in the
animals are determined ultimately by the circulating levels of
mutated hormones. Several different plasmids that encoded different
mutated amino acid sequences of GHRH or functional biological
equivalent thereof are as follows:
TABLE-US-00004 Plasmid Encoded Amino Acid Sequence wt-GHRH
YADAIFTNSYRKVLGQLSARKLLQDIMSRQQGERNQEQGA-OH (SeqID No: 10) HV-GHRH
HVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SeqID No: 1) TI-GHRH
YIDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SeqID No: 2) TV-GHRH
YVDAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SeqID No: 3)
15/27/28-GHRH YADAIFTNSYRKVLAQLSARKLLQDILNRQQGERNQEQGA-OH (SeqID
No: 4)
[0308] In general, the encoded GHRH or functional biological
equivalent thereof is of formula:
TABLE-US-00005 (SeqID No: 6)
-X.sub.1-X.sub.2-DAIFTNSYRKVL-X.sub.3-QLSARKLLQDI-X.sub.4-X.sub.5-
RQQGERNQEQGA-OH
wherein: X.sub.1 is a D- or L-isomer of an amino acid selected from
the group consisting of tyrosine ("Y"), or histidine ("H"); X.sub.2
is a D- or L-isomer of an amino acid selected from the group
consisting of alanine ("A"), valine ("V"), or isoleucine ("I");
X.sub.3 is a D- or L-isomer of an amino acid selected from the
group consisting of alanine ("A") or glycine ("G"); X.sub.4 is a D-
or L-isomer of an amino acid selected from the group consisting of
methionine ("M"), or leucine ("L"); X.sub.5 is a D- or L-isomer of
an amino acid selected from the group consisting of serine ("S") or
asparagine ("N").
[0309] Another plasmid that was utilized included the pSP-SEAP
construct (SeqID No: 16) that contains the SacI/HindIII SPc5-12
fragment, SEAP gene and SV40 3'UTR from pSEAP-2 Basic Vector
(Clontech Laboratories, Inc.; Palo Alto, Calif.).
[0310] The plasmids described above do not contain polylinker,
IGF-I gene, a skeletal alpha-actin promoter or a skeletal alpha
actin 3' UTR/NCR. Furthermore, these plasmids were introduced by
muscle injection, followed by in vivo electroporation, as described
below.
[0311] In terms of "functional biological equivalents", it is well
understood by the skilled artisan that, inherent in the definition
of a "biologically functional equivalent" protein, polypeptide,
and/or polynucleotide, is the concept that there is a limit to the
number of changes that may be made within a defined portion of the
molecule while retaining a molecule with an acceptable level of
equivalent biological activity. Functional biological equivalents
are thus defined herein as those proteins (and polynucleotides) in
selected amino acids (or codons) that may be substituted. A peptide
comprising a functional biological equivalent of GHRH is a
polypeptide that has been engineered to contain distinct amino acid
sequences while simultaneously having similar or improved
biologically activity when compared to GHRH. For example, one
biological activity of GHRH is to facilitate growth hormone ("GH")
secretion in the subject.
[0312] Electroporation devices. A BTX T820 generator (BTX, division
of Genetronics Inc., CA) was used to deliver square wave pulses in
all experiments. Voltage conditions of 100-200 V/cm, 6 pulses, 60
milliseconds per pulse were used. Caliper and needle electrodes
(BTX, division of Genetronics Inc., CA) were used to deliver in
vivo electric pulses. The plate (caliper) electrodes consisted of
1.5 cm square metallic blocks mounted on a ruler, so the distance
between the plates could be easily assessed; the 6-needle device
consists of a circular array (1 cm diameter) of six equally spaced
filled 21-gauge needles mounted on a non-conductive material. The
3-needle device consists of two filled and one cannular needle, the
last one being used both as an electrode and to deliver the
plasmid. All needles were 2 cm in length. In all injections the
needles were completely inserted into the muscle.
[0313] A skilled artisan recognizes that any similar
electroporation device and parameters may be used in the present
invention so long as the device delivers the nucleic acid sequence
to the cell, tissue, or organism.
[0314] Intramuscular injection of plasmid DNA in porcine. Two- to
three-week-old hybrid barrows
(Yorkshire.times.Landrace.times.Hampshire.times.Duroc)(Huntsville,
Tex.), 4-5 kg in weight, or Yorkshire.times.Landrace pigs were used
in the secreted embryonic alkaline phosphatase studies (n=3/group).
For the GHRH plasmid studies, time-pregnant sows
(Yorkshire.times.Landrace) were brought three weeks before the
scheduled parturition date to the Children Nutrition Research
Center at Baylor College of Medicine. Piglets were born in the
facility. Piglets were assigned randomly to one of the experimental
(n=2 pigs/group/series) or controls (n=3) groups. All experiments
were repeated three times. The animals were suckled for the first
21 days and then individually housed with ad-lib access to water.
For GHRH studies, after weaning, pigs were fed a 24% protein diet
(Producers Cooperative Association, Bryan, Tex.) at 6% of their
body weight daily. The animals were weighed twice a week, at the
same time of day, and the amount of feed was subsequently
determined. Animals were maintained in accordance with NIH Guide,
USDA and Animal Welfare Act guidelines, and approved by the Baylor
College of Medicine IACUC.
[0315] Endotoxin-free plasmid (Qiagen Inc., Chatsworth, Calif.,
USA) preparations were diluted in PBS, pH 7.4 to 1 mg/ml. Plasmid
DNA was injected through the intact skin into the semitendinosus or
the longissimus dorsi muscle using a 21 g needle. Two minutes
later, external caliper electrodes or injectable electrodes
(6-needle array or 3-needle array) were applied to the injected
muscle, and 6 pulses of 200V/cm, 60 millisecond/pulse were applied.
The polarity of the pulses was either constant or inverted between
the needles.
[0316] Blood was collected by jugular puncture before plasmid
injection, and at 3, 7, 14, 21, 35 and 45 days post-injection. At
50 days post-injection, animals were sacrificed and internal organs
and the injected muscle were collected, weighed, frozen in liquid
nitrogen, and stored at -80.degree. C., or placed in 10% buffered
formalin for histology.
[0317] Although in vivo electroporation is the preferred method for
delivering the nucleic acid constructs into the cells of the
subject, suitable methods for nucleic acid delivery for
transformation of an organelle, a cell, a tissue or an organism for
use with the current invention are believed to include virtually
any method by which a nucleic acid (e.g., DNA) can be introduced
into an organelle, a cell, a tissue or an organism, as described
herein or as would be known to one of ordinary skill in the art.
Such methods include, but are not limited to, direct delivery of
DNA such as by ex vivo transfection (Nabel et al., 1989; Wilson et
al., 1989), by injection (U.S. Pat. Nos. 5,994,624, 5,981,274,
5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466
and 5,580,859, each incorporated herein by reference), including
microinjection (Harland and Weintraub, 1985) U.S. Pat. No.
5,789,215, incorporated herein by reference); by electroporation
(U.S. Pat. No. 5,384,253, incorporated herein by reference; (Potter
et al., 1984; Tur-Kaspa et al., 1986); by calcium phosphate
precipitation (Chen and Okayama, 1987; Graham and van der Eb, 1973;
Rippe et al., 1990); by using DEAE-dextran followed by polyethylene
glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al.,
1987); by liposome mediated transfection (Hafez et al., 2001; Hamm
et al., 2002; Madry et al., 2001; Raghavachari and Fahl, 2002;
Wiethoff et al., 2001) and receptor-mediated transfection (Wu and
Wu, 1988a; Wu and Wu, 1988b); by microprojectile bombardment (PCT
Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos.
5,610,042; 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880,
and each incorporated herein by reference); by agitation with
silicon carbide fibers ((Johnson et al., 1992); U.S. Pat. Nos.
5,302,523 and 5,464,765, each incorporated herein by reference); by
Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,591,616 and
5,563,055, each incorporated herein by reference); by PEG-mediated
transformation of protoplasts (Omirulleh et al., 1993); U.S. Pat.
Nos. 4,684,611 and 4,952,500, each incorporated herein by
reference); by desiccation/inhibition-mediated DNA uptake (Potrykus
et al., 1985), and any combination of such methods. Through the
application of techniques such as these, organelle(s), cell(s),
tissue(s) or organism(s) may be stably or transiently
transformed.
[0318] Porcine plasma IGF-I and insulin concentrations. Porcine
IGF-I was measured by heterologous human radioimmunometric assay
(Diagnostic System Lab., Webster, Tex.). The sensitivity of the
assay was 0.8 ng/ml; intra-assay and inter-assay variation was 3.4%
and 4.5%, respectively. Porcine insulin was measured using a
heterologous human radioimmunoassay (Linco Research Inc., St.
Charles, Mo.). The sensitivity level of the assay was 2
.quadrature.U/ml; intra-assay and inter-assay variation was 3.2%
and 3.9% respectively.
[0319] Body composition data. Weights were measured on the same
calibrated scales (certified to have an accuracy to .+-.0.2 kg and
a coefficient of variation of 0.3%) throughout the study, twice a
week. Body composition measurements were performed in vivo, 50 days
after birth. The piglets were anesthetized using a combination of
xylazine (15 mg/kg) and ketamine (2 mg/kg) and the total body
content of fat, percent of fat, non-bone lean tissue mass and bone
mineral content was measured by dual-energy x-ray absorptiometry
(Hologic QDR-2000, Waltham, Mass.) ("DEXA") (Toner et al., 1996).
Total body potassium was measured in a potassium chamber ("K40")
using a whole body detector (Cohn et al., 1984).
[0320] Statistics Data are analyzed using STATISTICA analysis
package (StatSoft, Inc. Tulsa, Okla.). Values shown in the figures
are the mean.+-.s.e.m. Specific P values were obtained by
comparison using ANOVA. A P<0.05 was set as the level of
statistical significance.
Example 2
Constitutive GHRH System In Vivo
[0321] To test the constitutive GHRH system in vivo, 7.5 micrograms
of pSP-GHRH or functional biological equivalents (FIG. 1) were
delivered into SCID mice. All GHRH analog sequences were obtained
by site directed mutagenesis of the porcine wild type sequence.
Groups of five mice were injected with 7.5 micrograms of plasmid
expressing either one of the GHRH analogs, or a
pSP-beta-galactosidase as control. At 45 days post-injection,
animals were analyzed by PIXImus (Draghia-Akli et al., 2002)(DEXA
for mice), sacrificed, blood and organs were collected and weighed.
At the end of the experiment, the TI-GHRH and HV-GHRH animals were
significantly bigger than controls (FIG. 2). The body composition
of the injected SCID mice was also altered. At 45 days
post-injection, animals that were injected with the TI mutant had a
significant increase in lean body mass of 11% versus controls,
p<0.036. The HV-GHRH injected animals had a significant increase
of the lean body mass of almost 5% (FIG. 3). All GHRH injected
groups had larger bone areas than the control animals, up to 10.7%,
in the TI-GHRH injected group, p<0.027. (FIG. 4). At 14 and 28
days post-injection, blood was collected and IGF-I levels were
measured (FIG. 5). All GHRH injected groups had significantly
increased IGF-I levels compared with control animals, up to
p<0.005. Some groups developed neutralizing antibodies, and in
these cases the IGF-I levels dropped at the second time point. The
animals injected with TI-GHRH did not develop any antibodies, and
their GHRH expression continued to 45 days, correlating with
significant changes in their body composition.
Example 3
Inducible GHRH System In Vitro
[0322] To test the inducible GHRH system in vitro, primary chicken
myoblasts were transfected as described previously (Bergsma et al.,
1986; Draghia-Akli et al., 1997) with 4 micrograms of a mixture of
the GHRH/GeneSwitch.RTM. plasmids, pGR1774 (inducible GHRH)/pGS1633
(Gene Switch.RTM.) in a 10:1 w/w ratio, which gave the best overall
expression in skeletal muscle cells, and cells were allowed to
differentiate into post-mitotic myotubes. At 24 and 48 hours after
transfection, cells were washed in PBS, and MFP was added, where
indicated, to the culture media. Media and cells were harvested 72
hours post-differentiation. 20 .mu.g of total RNA was DNase I
treated, size separated in 1.5% agarose-formaldehyde gel and
transferred to nylon membrane. The membranes were hybridized with a
specific GHRH cDNA probe .sup.32P-labeled by random priming.
Negative controls were cells transfected by the GeneSwitch.RTM. and
GHRH plasmids, but not treated with MFP, or cells transfected by
the inducible GHRH plasmid alone. The positive control was cells
transfected by a constitutively expressed GHRH plasmid that was
driven by a synthetic muscle-specific promoter ("SP-GHRH"). GHRH
transcripts of the expected size of 0.35 kb were only observed in
cells transfected with the GeneSwitch.RTM./inducible GHRH plasmids
and treated with MFP, and in cells transfected with the positive
control (FIG. 7). No GHRH transcripts were detected in cells not
treated with MFP or in cells transfected by the inducible GHRH
plasmid alone.
Example 4
GHRH/Geneswitch.RTM. System In Vivo-Improved Body Composition and
Fat Body Mass/Total Weight
[0323] For the in vivo experiments, the plasmids for the
GHRH/GeneSwitch.RTM. system were delivered to the muscles of SCID
mice. The left tibialis anterior muscle was injected with 10 .mu.g
of a 10:1 mixture of pGR1774/pGS1633, followed by caliper
electroporation (Draghia-Akli et al., 1999). At twenty-one days
post-injection, animals were injected inter perineum ("i.p.") with
250 micrograms/kg of MFP for 3 days. On the fourth day, the animals
were bled and serum was used to measure IGF-I levels. Mouse IGF-I
was measured by heterologous, 100% cross-reacting rat
radioimmunoassay. The sensitivity of the assay was 0.8 ng/ml;
intra-assay and inter-assay variation was 3.4% and 4.5%
respectively. Following administration of MFP for 4 consecutive
days, IGF-I levels increased from 1100.86.+-.33.67 ng/ml to
1797.28.+-.164.96 ng/ml (p<0.0005). Significant changes in the
IGF-I levels were seen when the MFP group was compared with the
control group 1086.78.+-.65.34 ng/ml, p<0.0006 (animals that
received a control beta-galactosidase plasmid), 1171.79.+-.42
ng/ml, p<0.001 (animals that received the GHRH/GeneSwitch.RTM.
plasmids but were not dosed with MFP). Upon repeated administration
of MFP to the animals using the same protocol followed by recovery
to background 7 days over 149 days, serum IGF-I levels rose
repeatedly 1.1-1.7 fold over the uninjected controls (FIG. 8).
Animals induced with MFP had statistically significant higher IGF-I
levels.
[0324] Body weight was similar for all of the groups during the
first 125 days of the study (FIG. 9). However, from day 125 to day
149, mice were dosed with MFP every day. A 7.5% increased body
weight was observed in the chronically MFP-induced
GHRH/GeneSwitch.RTM. animals, averaging 31.84.+-.0.12 g
(p<0.027), compared with C-gal controls, 29.62.+-.0.98 g, and
with animals that were not induced with MFP, 30.53.+-.0.59 g. All
values are average.+-.SEM. Organs (lungs, heart, liver, kidney,
stomach, intestine, adrenals, gonads, brain) were collected and
weighed. No organomegaly or associated pathology was observed in
any of the animals. Pituitary glands were dissected within the
first minutes post-mortem, and weighed. Pituitary weight/total body
weight (FIG. 10) increased upon chronic stimulation of the
GHRH/GeneSwitch.RTM. by 20% (7.35.+-.0.31.times.10.sup.-5),
compared with .quadrature.-gal controls
(6.13.+-.0.46.times.10.sup.-5), and animals not dosed with MFP
(6.3.+-.0.22.times.10.sup.-5), p<0.035. There was no
significantly statistical difference between the .quadrature.-gal
injected animals and animals that were injected with the
GHRH/GeneSwitch.RTM. system, but not given MFP. Although not to be
bound by theory, the increase in pituitary weight was probably due
to somatotrophs hypertrophy, as it is known that GHRH is capable of
stimulating the synthesis/secretion of GH from the anterior
pituitary and has a specific hypertrophic effect on somatotrophs
(Morel et al., 1999; Murray et al., 2000). At the end of the
experiment, body composition was analyzed in vivo, by dual-energy
x-ray absorptiometry ("DEXA"), using a high resolution PIXImus
scanner. Body composition studies by PIXImus (total body fat,
non-bone lean tissue mass and bone mineral area, content and
density) showed significant changes in chronically MFP induced
animals injected with the GHRH/GeneSwitch.RTM. system. Lean body
mass (non-bone) (FIG. 11) increased by 2.5% in GHRH/GeneSwitch.RTM.
animals+MFP (87.44.+-.0.65%, versus .quadrature.-gal 84.94.+-.0.6%,
and no MFP animals 84.88.+-.0.3%), p<0.022. Fat mass (FIG. 12)
decreased by 2% in GHRH/GeneSwitch.RTM. animals (12.59.+-.0.62%,
versus .quadrature.-gal 14.57.+-.0.75%, and no MFP animals
15.09.+-.0.3%), p<0.05.
Example 5
GHRH/Geneswitch.RTM. In Vivo-Increased Bone Area and Mineral
Content
[0325] One aspect of the present invention involves demonstrating
that the introduction of mifepristone-inducible heterologous
nucleic acid sequences encoding GHRH or functional biological
equivalent thereof into the cells of subjects can lead to high
levels of circulating hormones (Mir et al., 1999), without the
disadvantages (e.g. high production costs, safety concerns with the
virus backbone, or ex vivo manipulation) associated with viral
vector delivery or organoids (Barr and Leiden, 1991; Dhawan et al.,
1991; Draghia-Akli et al., 1999). In addition, the invention must
demonstrate that animal growth and body composition can be
efficiently regulated by mifepristone following in vivo
electroporation of the GeneSwitch.RTM. technology (i.e.
mifepristone-inducible heterologous nucleic acid sequences encoding
GHRH or functional biological equivalent thereof) into skeletal
muscle of the subject, as schematically diagrammed in FIG. 6.
Enhanced biological potency, delivery and proper gene expression
regulation was observed over 149 days post-injection, and
effectively reduced the theoretical quantity of GHRH needed to
achieve physiological levels of GH secretion when compared to the
recombinant GHRH therapies. Post-injected subjects did not
experience any side effects from the GeneSwitch.RTM. technology
therapy. For example, mice had normal biochemical profiles, and no
associated pathology or organomegaly. From a functional standpoint,
the IGF-I levels increased, growth was enhanced by 7.5%, and
changes in body composition (e.g. with increased lean body mass by
2.5% and decreased fat by 2%) were observed following chronic
induction of the GHRH/Gene Switch system. In addition, bone mineral
density increased by 6%, and the stimulation of GHRH on bone
metabolism were even more remarkable. Although not to be bound by
theory, the observed pituitary hypertrophy was indicative that
ectopic expression of myogenic GHRH plasmids operates through the
natural GH axis (stimulation of GH synthesis and secretion at the
pituitary level). This long-lasting regulated therapy has the
potential to replace classical GH therapy regimens and may
stimulate the GH axis in a more physiologically appropriate manner.
It is known that GHRH stimulates bone formation (Dubreuil et al.,
1996), and the described GeneSwitch.RTM. therapy may be used to
promote post-fracture bone growth.
[0326] Upon chronic stimulation of the GHRH/GeneSwitch.RTM. system,
significant changes occurred in bone area (FIG. 13), that increased
by 7%, (12.811.+-.0.14 cm.sup.2, versus B=Beta .quadrature.-gal
injected controls 11.98.+-.0.3 cm.sup.2, or no MFP animals
12.07.+-.0.26 cm.sup.2), p<0.0006, bone mineral content (FIG.
14) increased by 14.6% (0.755.+-.0.012 g, versus .quadrature.-gal
injected controls 0.659.+-.0.019 g, or no MFP animals
0.694.+-.0.023 cm.sup.2), p<0.002, and bone mineral density
increased by 6% (0.059.+-.0.0007 g/cm.sup.2, versus
.quadrature.-gal injected controls 0.056.+-.0.0009 g/cm.sup.2, or
no MFP animals 0.057.+-.0.0007 g/cm.sup.2), p<0.012.
Practically, there is no overall difference between the
.quadrature.-gal injected animals and animals that were injected
with the GHRH/GeneSwitch.RTM., but were not given MFP, which
supports the absence of GHRH expression by the GHRH/GeneSwitch.RTM.
plasmids in the absence of MFP dosing.
Example 6
Low Voltage Electroporation Increases Plasmid Uptake and Expression
in Adult Pigs
[0327] Direct intra-muscular plasmid DNA injection followed by
electroporation is a method for the local and controlled delivery
of plasmid DNA into skeletal muscle. It has the advantage that is
uses low plasmid quantities (as low as 0.1 mg in pigs), rather than
the high quantities typically used with passive delivery
modalities. Although not wanting to be bound by theory, the
mechanism of the increased plasmid uptake by electroporation
probably occurs through newly created membrane pores with or
without protein active transport. It has been shown that the degree
of permeabilization of the muscle cells is dependent on the
electric field intensity, length of pulses, shape and type of
electrodes (Bureau et al., 2000; Gilbert et al., 1997), and cell
size (Somiari et al., 2000). Classical electrode configuration,
plates or a pair of wire electrodes placed 4 mm apart were shown to
be effective in rodents, but in large mammals as pigs or humans the
increased resistance of the skin, the thickness of the subcutaneous
fat tissue, and the concern for tissue damage if the intensity of
the electric field would be proportionally increased, make these
types of electrodes unpractical. The porcine muscle fibers are
quite large and consequently more suitable for
electropermeabilization than rodent muscle. Data provided herein
indicate that a single injection of an optimum dosage of plasmid
followed by electroporation with intramuscular applicators is
sufficient to produce therapeutic plasma hormone levels in a large
mammal with biologically significant effects on the body fat
distribution and lean body mass of the subject.
[0328] External caliper electrodes and injectable electrodes were
evaluated to determine the type of electrode needed to achieve a
physiologically relevant level of a secreted reporter protein in
4-5 kg hybrid pigs. Reporter vectors expressing secreted embryonic
alkaline phosphatase ("SEAP") were used in these studies at a dose
of 2 mg pSP-SEAP/animal. Six-needle and 3-needle array electrodes
were compared with standard caliper electrodes (FIG. 15).
Conditions of 6 pulses, 200V/cm, 60 milliseconds/pulse, previously
tested as being the most effective in pigs (Draghia-Akli et al.,
1999) were applied in all tests. For the three-needle electrode,
three pulses were applied in one direction, then the polarity was
changed and the next three pulses were delivered in the opposite
direction. SEAP values were measured at day 0, day 3 and day 7
post-injection. Seven days post-injection, the SEAP levels were
9.33.+-.2.26 ng/(mlkg) in plasmid-injected and caliper
electroporated animals, compared to 0.02.+-.0.005 ng/(mlkg) in
vehicle-injected animals. Using the 3-needle and 6-needle arrays, a
12.4 and 19 fold increase in SEAP values was obtained compared to
caliper delivery (116.07.+-.44.36 ng/(mlkg), and 177.41.+-.18.44
ng/(mlkg), respectively). When using the same number of pulses, but
lower voltage (100V/cm), and the 6-needle electrodes, the average
SEAP increased to 144.64.+-.11.82 ng/(mlkg) after seven days. When
longissimus dorsi and semitendinosus muscles were injected using
similar conditions, expression in the semitendinosus muscle was
slightly higher. Skin and muscle from the injected pigs were
collected at the end of the experiment (at 50 days post-injection)
and histologically analyzed. At 100-200 V/cm used in the injectable
electrodes experiments, no skin or muscle damage was seen for any
of the needle-type electrodes used.
Example 7
Increased Efficiency Using Needle-Type Electroporation Delivery for
Therapeutic Proteins
[0329] Not wanting to be bound by theory, growth hormone releasing
hormone ("GHRH") stimulates the production and release from the
anterior pituitary of growth hormone ("GH"), which in turn
stimulates the production of IGF-I from the liver and other target
organs (Frohman et al., 1968). In previous studies (Draghia-Akli et
al., 1999), young pigs weighing 4-5 kg, were injected with 10 mg
myogenic vector expressing a mutated form of GHRH, stable to
proteases ("pSP-HV-GHRH") and electroporated using a caliper
electrode.
[0330] The present invention involves determination of the best age
for treatment of young pigs. Groups of 2 piglets were injected with
2 mg pSP-HV-GHRH using the 6-needle array electrodes at different
time points: birth, 7, 14 and 21 days of age (FIG. 16). Each animal
received one injection. The group injected at 14 days of age
demonstrated the best weight gain, (statistically significant and
different from PBS controls (n=3) at every time point (final
weights: 25.8.+-.1.5 kg versus 19.7.+-.0.03 kg, p<0.013)). The
next best group was injected at 7 days of age, and weighed
21.9.+-.1.5 kg at age 50 days, p<0.02.
[0331] In a parallel study, the reduction in the plasmid quantity
needed to achieve improved growth and changes in the metabolic and
hormonal profile of pigs was explored. Groups of two piglets each
(Yorkshire.times.Landrace) were injected at 10 days of age with
pSP-HV-GHRH (3 mg, 1 mg, 0.1 mg), and electroporated using a
6-needle array electrode (FIG. 17). The group injected with 0.1 mg
of plasmid had the greater weight gain, with statistically
significant differences to controls (n=3) to 50 days of age
(22.4.+-.0.8 kg versus 19.7.+-.0.03 kg, p<0.012). One animal in
the group injected at 21 days and one animal injected with the
highest plasmid dose (3 mg) developed neutralizing antibodies
against the mutated HV-GHRH and showed significant reduced rates of
weight gain (at 50 days post-injection 15.6 kg and 15.95 kg,
respectively, versus more than 21 kg for the paired animal in the
same treatment group). No other group developed neutralizing
antibodies. Thus, the minimal plasmid dosage (0.1 mg) and injection
at optimum age using the 6-needle electrodes resulted in the best
growth performances. It is noteworthy that in previous studies the
inventors used 100-fold less, i.e., 10 mg pSP-HV-GHRH with the
caliper electrodes to produce similar changes.
[0332] An indication of increased systemic levels of GHRH and GH is
an increase in serum IGF-I concentration. The level of serum IGF-I
started to rise at 3 days post-injection in pigs that received the
0.1 and 1 mg doses of pSP-HV-GHRH (FIG. 18). By 35 days after the
injection (age of animals: 45 days), serum IGF-I concentrations
were approximately 10-fold higher in pigs injected with 0.1 mg and
7-fold higher in pigs injected with 1 mg plasmid compared with
controls (p<0.007 and p<0.04 respectively).
[0333] In pSP-HV-GHRH injected pigs, under optimum conditions
(Table 1) serum urea decreased (8.36.+-.1.33 to 9.67.+-.1.27 mg/ml
in pSP-HV-GHRH injected pigs versus 11.14.+-.1.9 mg/ml in controls,
respectively (p<0.05), indicating decreased amino acid
catabolism. Serum glucose levels were similar between the plasmid
pSP-HV-GHRH injected pigs and controls; insulin levels were normal
and within the control range.
TABLE-US-00006 TABLE 1 The plasma metabolic profile of pSP-HV-GHRH
injected and control pigs. Glucose Urea Creatine Total Protein
(mg/ml) (mg/ml) (mg/ml) (g/dl) Group Age Day 0 125.82 .+-. 5.64
8.36 .+-. 1.33 0.85 .+-. 0.05 4.81 .+-. 0.11 p < 0.01 Day 7
122.43 .+-. 5.05 9.43 .+-. 1.67 0.87 .+-. 0.04 5.21 .+-. 0.19 p
< 0.03 Day 14 129.54 .+-. 6.39 9.62 .+-. 1.72 1.00 .+-. 0.04
5.22 .+-. 0.20 p < 0.02 Day 21 110.25 .+-. 5.02 13.83 .+-. 1.2
0.93 .+-. 0.05 4.81 .+-. 0.21 Dose 3 mg 111.07 .+-. 3.88 10.50 .+-.
1.87 0.9 .+-. 0.09 3.89 .+-. 0.16 p < 0.05 1 mg 121.63 .+-. 2.93
9.44 .+-. 1.07 0.81 .+-. 0.06 4.11 .+-. 0.11 p < 0.02 0.1 mg
120.73 .+-. 2.53 9.67 .+-. 1.27 0.95 .+-. 0.05 4.05 .+-. 0.19 p
< 0.02 Control 119.77 .+-. 3.67 12.81 .+-. 2.01 0.98 .+-. 0.08
4.00 .+-. 0.11
[0334] The fact that these animals have a normal carbohydrate
metabolism is very important, as most livestock and/or patients
under recombinant GH therapy develop impaired glucose metabolism
and insulin resistance. Creatinine concentration (a measure of
kidney function) was normal in all animals. Pigs that developed
antibodies to GHRH showed a tendency to increased urea levels and
decreased glucose levels.
[0335] Body composition studies by dual-energy x-ray absorptiometry
(total body fat, non-bone lean tissue mass and bone mineral
content), K40 potassium (lean body mass) and carcass neutron
activation analysis (nitrogen) showed a proportional increase of
all internal organs in GHRH injected animals (heart, lung, liver,
spleen, brain, adrenals, stomach, kidney, pancreas, intestine).
Nevertheless, the final body composition was different: animals
injected with pSP-HV-GHRH at different ages gained proportionally
less fat than controls and were leaner at the end of the study
(4.34.+-.0.04 g of fat gained/kg of fat free mass gained per day
for injection at birth, 4.4.+-.0.04 g for injection at 7 days,
versus controls 5.63.+-.0.34 g, p<0.05). Bone mineral density
was higher in animals injected at 14 days after birth, and
correlates with increased efficacy of the treatment: 0.363.+-.0.005
g/cm.sup.2 versus 0.329.+-.0.003 g/cm.sup.2 in controls,
p<0.004.
[0336] Treated pigs did not experience any side effects from the
therapy, had normal biochemical profiles, and had no associated
pathology or organomegaly. From a functional standpoint, the
increases in IGF-I levels and enhancement in growth and changes in
body composition (with decreased fat deposition by 22%) were
dramatic in extent. The effects of the stimulation of GHRH on bone
metabolism were even more remarkable, with an increase in bone
mineral density by 10%. These results indicate that ectopic
expression of myogenic HV-GHRH vectors has the potential to replace
classical GH therapy regimens and may stimulate the GH axis in a
more physiologically appropriate manner. The HV-GHRH molecule,
which displays a high degree of stability and GH secretory activity
in pigs, may also be useful in human clinical medicine. However, a
skilled artisan is aware that a minimal plasmid dose should be
determined on a pertinent model and used in order to avoid the
unwanted pathology associated with antibody development, and
routine methods in the art and/or described herein teach how to
achieve this goal.
[0337] The molecular techniques used to produce alterations in any
conceivable encoded nucleic acid sequences are well established,
and exemplified by the large number of scientific publications and
patents in the field of molecular biology. Despite the accuracy of
the molecular techniques used to create distinctive nucleic acid
sequences, a skilled artisan recognizes that the expression of any
given nucleic acid will influence the complex biochemistry of an
entire organism. Thus, the highly predictable nature of
constructing unique nucleic acid sequences must not be confused
with unknown facts of an associated biological effect.
[0338] The invention described herein involves the utilization of
several distinctive GHRH or analog nucleic acid sequences. Based
upon the current understanding of protein-protein interactions, it
is neither obvious nor possible to accurately speculate upon the in
vivo parameters (e.g. half life, efficacy, post-translational
modifications, etc.) of a GHRH sequence that contains a point
mutation which alters a single amino acid in the polypeptide chain.
As seen in the Examples provided herein, mutation of a few base
pairs gave rise to GHRH mutants with significantly longer
bio-availability. The endogenous GHRH has a half-life of 6-12
minutes in different species. The HV-GHRH has a half-life of 6
hours. In further analysis, the TI-GHRH (that has only two base
pair difference with the HV-GHRH) has been shown to have a much
higher effect in vivo on lean body mass than the HV-GHRH (from
simple to double). This property was not evident in extensive in
vitro studies on pituitary cell. Correspondingly, one skilled in
the art would know how to perform the plasmid-mediated
supplementation of GHRH or the related recombinant protein
experimentation(s), characterizing variations and permutations on a
unique nucleic acid sequence in a specific tissue to accurately
evaluate the in vivo effect within a living organism. Therefore,
the utilization of the distinctive nucleic acid sequence encoding
GHRH or functional biological equivalent thereof or corresponding
recombinant protein as a method to decrease body fat proportion and
increase lean body mass could not have been predicted based on
speculation.
[0339] Although not wanting to be bound by theory, it is believed
that an increase in GHRH or functional biological equivalent will
increase the GH levels to decrease body fat proportion and increase
lean body mass. Hormones (e.g. GHRH and GH) often contain a complex
feedback-regulated pathway. Without direct experimentation of GHRH
or analogs used in gene or recombinant protein therapy, it could
not have been predicted by one skilled in the art to determine
which concentrations of non-native encoded sequences will yield
desired results. Ideal regulation of a nucleic acid sequence
encoding GHRH or functional biological equivalent thereof is
further complicated by the tissue used for polynucleotide delivery,
and would not have been obvious to one skilled in the art without
actual experimentation with the distinctive sequence in a
particular tissue. The invention described herein contains the
descriptions and results of essential experimentation that explored
tissue specific and inducible regulation of distinctive nucleic
acid sequences that encoded GHRH or functional biological
equivalent thereof, which was not obvious based upon prior art. The
present invention is a significant step forward in developing
non-viral therapy for large animals, including humans. In order for
gene therapies to be transferred from rodents to large mammals, and
ultimately to humans, it was surprising that extremely low
quantities of plasmid were effective. It is shown herein that as
little as 0.1 mg plasmid delivered under the proper electroporation
conditions had an important biological impact that decreases the
body fat proportion, increases lean body mass ("LBM"), or both of a
subject. This plasmid quantity was 100 fold lower than the
theoretical one, and could not have been predicted from the
relative doses used in rodents (in average 1 mg/kg). Although not
wanting to be bound by theory, unlike other therapies using growth
factors (GH and/or IGF-I), GHRH is stimulating the endogenous
secretion of hormones, and enhancing the own bio-potential of the
animal, with no adverse effects. This experimental finding cannot
be theoretically predicted, as the three hormones are members of
the same growth axis.
[0340] The increase in lean body mass, decrease in body fat
proportions, increase in bone density, and/or increase in bone rate
of healing are a consequence of the GHRH molecules present in the
subjects circulation, regardless of the means of the delivery. For
example, one would obtain the same effect by delivering appropriate
quantities of GHRH or analog thereof, outlined in FIG. 1, by
classical recombinant protein therapy or nucleic acid transfer.
Accordingly, successful plasmid-mediated supplementation of GHRH
requires accurate delivery of the encoded sequences to the cells of
a subject, resulting in expression of the gene product at levels
appropriate to produce a biological effect. The duration of
treatment will extend through the course of the disease symptoms,
and possibly continuously. Since the method to deliver nucleic acid
sequences to the cells of a subject is highly dependent on specific
diseases and the encoded gene, it could not have been predicted by
one skilled in the art which method and conditions are appropriate
without laborious and failed experimentations. Thus, the preferred
method of outlined for this invention is in vivo
electroporation.
[0341] One skilled in the art readily appreciates that the present
invention is well adapted to carry out the objectives and obtain
the ends and advantages mentioned as well as those inherent
therein. Methods, procedures, techniques, and kits 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 invention.
[0342] All of the methods and compositions disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the methods and compositions
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the systems and compositions without
departing from the concept, spirit, and scope of the invention.
More specifically, it will be apparent that certain agents which
are both chemically, structurally and physiologically related may
be substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope, and concept of the invention as defined
by the appended claims.
Sequence CWU 1
1
50140PRTartificial sequenceThis is a functional biological
equivalent of GHRH. 1His Val Asp Ala Ile Phe Thr Asn Ser Tyr Arg
Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile
Leu Asn Arg Gln Gln Gly 20 25 30Glu Arg Asn Gln Glu Gln Gly Ala 35
40240PRTartificial sequenceThis is a functional biological
equivalent of GHRH. 2Tyr Ile Asp Ala Ile Phe Thr Asn Ser Tyr Arg
Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile
Leu Asn Arg Gln Gln Gly 20 25 30Glu Arg Asn Gln Glu Gln Gly Ala 35
40340PRTartificial sequenceThis is a functional biological
equivalent of GHRH. 3Tyr Val Asp Ala Ile Phe Thr Asn Ser Tyr Arg
Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile
Leu Asn Arg Gln Gln Gly 20 25 30Glu Arg Asn Gln Glu Gln Gly Ala 35
40440PRTartificial sequenceThis is a functional biological
equivalent of GHRH. 4Tyr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg
Lys Val Leu Ala Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile
Leu Asn Arg Gln Gln Gly 20 25 30Glu Arg Asn Gln Glu Gln Gly Ala 35
40544PRTartificial sequenceThis is the artificial sequence for GHRH
(1-44)NH2 5Thr Ala Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val Leu
Gly Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Met Ser Arg
Gln Gln Gly 20 25 30Glu Ser Asn Gln Glu Arg Gly Ala Arg Ala Arg Leu
35 40640PRTartificial sequenceThis is the artificial sequence for
GHRH (1-40)OH. 6Xaa Xaa Asp Ala Ile Phe Thr Asn Ser Tyr Arg Lys Val
Leu Xaa Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln Asp Ile Xaa Xaa
Arg Gln Gln Gly 20 25 30Glu Arg Asn Gln Glu Gln Gly Ala 35
407323DNAartificial sequenceThis is a nucleic acid sequence of a
eukaryotic promoter c5-12. 7cggccgtccg ccctcggcac catcctcacg
acacccaaat atggcgacgg gtgaggaatg 60gtggggagtt atttttagag cggtgaggaa
ggtgggcagg cagcaggtgt tggcgctcta 120aaaataactc ccgggagtta
tttttagagc ggaggaatgg tggacaccca aatatggcga 180cggttcctca
cccgtcgcca tatttgggtg tccgccctcg gccggggccg cattcctggg
240ggccgggcgg tgctcccgcc cgcctcgata aaaggctccg gggccggcgg
cggcccacga 300gctacccgga ggagcgggag gcg 3238190DNAartificial
sequenceNucleic acid sequence of a hGH poly A tail. 8gggtggcatc
cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60gtgcccacca
gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
120ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
tgggaagaca 180acctgtaggg 1909219DNAartificial sequencecDNA for
Porcine GHRH 9atggtgctct gggtgttctt ctttgtgatc ctcaccctca
gcaacagctc ccactgctcc 60ccacctcccc ctttgaccct caggatgcgg cggcacgtag
atgccatctt caccaacagc 120taccggaagg tgctggccca gctgtccgcc
cgcaagctgc tccaggacat cctgaacagg 180cagcagggag agaggaacca
agagcaagga gcataatga 2191040PRTartificial sequenceamino acid
sequence for porcine GHRH. 10Tyr Ala Asp Ala Ile Phe Thr Asn Ser
Tyr Arg Lys Val Leu Gly Gln1 5 10 15Leu Ser Ala Arg Lys Leu Leu Gln
Asp Ile Met Ser Arg Gln Gln Gly 20 25 30Glu Arg Asn Gln Glu Gln Gly
Ala 35 40113534DNAartificial sequenceThe operatively linked
components of the HV-GHRH plasmid. 11gttgtaaaac gacggccagt
gaattgtaat acgactcact atagggcgaa ttggagctcc 60accgcggtgg cggccgtccg
ccctcggcac catcctcacg acacccaaat atggcgacgg 120gtgaggaatg
gtggggagtt atttttagag cggtgaggaa ggtgggcagg cagcaggtgt
180tggcgctcta aaaataactc ccgggagtta tttttagagc ggaggaatgg
tggacaccca 240aatatggcga cggttcctca cccgtcgcca tatttgggtg
tccgccctcg gccggggccg 300cattcctggg ggccgggcgg tgctcccgcc
cgcctcgata aaaggctccg gggccggcgg 360cggcccacga gctacccgga
ggagcgggag gcgccaagct ctagaactag tggatcccaa 420ggcccaactc
cccgaaccac tcagggtcct gtggacagct cacctagctg ccatggtgct
480ctgggtgttc ttctttgtga tcctcaccct cagcaacagc tcccactgct
ccccacctcc 540ccctttgacc ctcaggatgc ggcggcacgt agatgccatc
ttcaccaaca gctaccggaa 600ggtgctggcc cagctgtccg cccgcaagct
gctccaggac atcctgaaca ggcagcaggg 660agagaggaac caagagcaag
gagcataatg actgcaggaa ttcgatatca agcttatcgg 720ggtggcatcc
ctgtgacccc tccccagtgc ctctcctggc cctggaagtt gccactccag
780tgcccaccag ccttgtccta ataaaattaa gttgcatcat tttgtctgac
taggtgtcct 840tctataatat tatggggtgg aggggggtgg tatggagcaa
ggggcaagtt gggaagacaa 900cctgtagggc ctgcggggtc tattgggaac
caagctggag tgcagtggca caatcttggc 960tcactgcaat ctccgcctcc
tgggttcaag cgattctcct gcctcagcct cccgagttgt 1020tgggattcca
ggcatgcatg accaggctca gctaattttt gtttttttgg tagagacggg
1080gtttcaccat attggccagg ctggtctcca actcctaatc tcaggtgatc
tacccacctt 1140ggcctcccaa attgctggga ttacaggcgt gaaccactgc
tcccttccct gtccttctga 1200ttttaaaata actataccag caggaggacg
tccagacaca gcataggcta cctggccatg 1260cccaaccggt gggacatttg
agttgcttgc ttggcactgt cctctcatgc gttgggtcca 1320ctcagtagat
gcctgttgaa ttcgataccg tcgacctcga gggggggccc ggtaccagct
1380tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca
tagctgtttc 1440ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
acgagccgga agcataaagt 1500gtaaagcctg gggtgcctaa tgagtgagct
aactcacatt aattgcgttg cgctcactgc 1560ccgctttcca gtcgggaaac
ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 1620ggagaggcgg
tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
1680cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
cggttatcca 1740cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
aggccagcaa aaggccagga 1800accgtaaaaa ggccgcgttg ctggcgtttt
tccataggct ccgcccccct gacgagcatc 1860acaaaaatcg acgctcaagt
cagaggtggc gaaacccgac aggactataa agataccagg 1920cgtttccccc
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
1980acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
cgctgtaggt 2040atctcagttc ggtgtaggtc gttcgctcca agctgggctg
tgtgcacgaa ccccccgttc 2100agcccgaccg ctgcgcctta tccggtaact
atcgtcttga gtccaacccg gtaagacacg 2160acttatcgcc actggcagca
gccactggta acaggattag cagagcgagg tatgtaggcg 2220gtgctacaga
gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
2280gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
tcttgatccg 2340gcaaacaaac caccgctggt agcggtggtt tttttgtttg
caagcagcag attacgcgca 2400gaaaaaaagg atctcaagaa gatcctttga
tcttttctac ggggtctgac gctcagaaga 2460actcgtcaag aaggcgatag
aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa 2520gcacgaggaa
gcggtcagcc cattcgccgc caagctcttc agcaatatca cgggtagcca
2580acgctatgtc ctgatagcgg tccgccacac ccagccggcc acagtcgatg
aatccagaaa 2640agcggccatt ttccaccatg atattcggca agcaggcatc
gccatgggtc acgacgagat 2700cctcgccgtc gggcatgcgc gccttgagcc
tggcgaacag ttcggctggc gcgagcccct 2760gatgctcttc gtccagatca
tcctgatcga caagaccggc ttccatccga gtacgtgctc 2820gctcgatgcg
atgtttcgct tggtggtcga atgggcaggt agccggatca agcgtatgca
2880gccgccgcat tgcatcagcc atgatggata ctttctcggc aggagcaagg
tgagatgaca 2940ggagatcctg ccccggcact tcgcccaata gcagccagtc
ccttcccgct tcagtgacaa 3000cgtcgagcac agctgcgcaa ggaacgcccg
tcgtggccag ccacgatagc cgcgctgcct 3060cgtcctgcag ttcattcagg
gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc 3120cctgcgctga
cagccggaac acggcggcat cagagcagcc gattgtctgt tgtgcccagt
3180catagccgaa tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat
ccatcttgtt 3240caatcatgcg aaacgatcct catcctgtct cttgatcaga
tcttgatccc ctgcgccatc 3300agatccttgg cggcaagaaa gccatccagt
ttactttgca gggcttccca accttaccag 3360agggcgcccc agctggcaat
tccggttcgc ttgctgtcca taaaaccgcc cagtctagca 3420actgttggga
agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg
3480gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac
3534123534DNAartificial sequenceThe operatively linked components
of the TI-GHRH plasmid. 12gttgtaaaac gacggccagt gaattgtaat
acgactcact atagggcgaa ttggagctcc 60accgcggtgg cggccgtccg ccctcggcac
catcctcacg acacccaaat atggcgacgg 120gtgaggaatg gtggggagtt
atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180tggcgctcta
aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca
240aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg
gccggggccg 300cattcctggg ggccgggcgg tgctcccgcc cgcctcgata
aaaggctccg gggccggcgg 360cggcccacga gctacccgga ggagcgggag
gcgccaagct ctagaactag tggatcccaa 420ggcccaactc cccgaaccac
tcagggtcct gtggacagct cacctagctg ccatggtgct 480ctgggtgttc
ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc
540ccctttgacc ctcaggatgc ggcggtatat cgatgccatc ttcaccaaca
gctaccggaa 600ggtgctggcc cagctgtccg cccgcaagct gctccaggac
atcctgaaca ggcagcaggg 660agagaggaac caagagcaag gagcataatg
actgcaggaa ttcgatatca agcttatcgg 720ggtggcatcc ctgtgacccc
tccccagtgc ctctcctggc cctggaagtt gccactccag 780tgcccaccag
ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct
840tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
gggaagacaa 900cctgtagggc ctgcggggtc tattgggaac caagctggag
tgcagtggca caatcttggc 960tcactgcaat ctccgcctcc tgggttcaag
cgattctcct gcctcagcct cccgagttgt 1020tgggattcca ggcatgcatg
accaggctca gctaattttt gtttttttgg tagagacggg 1080gtttcaccat
attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt
1140ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
gtccttctga 1200ttttaaaata actataccag caggaggacg tccagacaca
gcataggcta cctggccatg 1260cccaaccggt gggacatttg agttgcttgc
ttggcactgt cctctcatgc gttgggtcca 1320ctcagtagat gcctgttgaa
ttcgataccg tcgacctcga gggggggccc ggtaccagct 1380tttgttccct
ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc
1440ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
agcataaagt 1500gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
aattgcgttg cgctcactgc 1560ccgctttcca gtcgggaaac ctgtcgtgcc
agctgcatta atgaatcggc caacgcgcgg 1620ggagaggcgg tttgcgtatt
gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1680cggtcgttcg
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
1740cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
aaggccagga 1800accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
ccgcccccct gacgagcatc 1860acaaaaatcg acgctcaagt cagaggtggc
gaaacccgac aggactataa agataccagg 1920cgtttccccc tggaagctcc
ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1980acctgtccgc
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
2040atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
ccccccgttc 2100agcccgaccg ctgcgcctta tccggtaact atcgtcttga
gtccaacccg gtaagacacg 2160acttatcgcc actggcagca gccactggta
acaggattag cagagcgagg tatgtaggcg 2220gtgctacaga gttcttgaag
tggtggccta actacggcta cactagaaga acagtatttg 2280gtatctgcgc
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
2340gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
attacgcgca 2400gaaaaaaagg atctcaagaa gatcctttga tcttttctac
ggggtctgac gctcagaaga 2460actcgtcaag aaggcgatag aaggcgatgc
gctgcgaatc gggagcggcg ataccgtaaa 2520gcacgaggaa gcggtcagcc
cattcgccgc caagctcttc agcaatatca cgggtagcca 2580acgctatgtc
ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
2640agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
acgacgagat 2700cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag
ttcggctggc gcgagcccct 2760gatgctcttc gtccagatca tcctgatcga
caagaccggc ttccatccga gtacgtgctc 2820gctcgatgcg atgtttcgct
tggtggtcga atgggcaggt agccggatca agcgtatgca 2880gccgccgcat
tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
2940ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
tcagtgacaa 3000cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag
ccacgatagc cgcgctgcct 3060cgtcctgcag ttcattcagg gcaccggaca
ggtcggtctt gacaaaaaga accgggcgcc 3120cctgcgctga cagccggaac
acggcggcat cagagcagcc gattgtctgt tgtgcccagt 3180catagccgaa
tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
3240caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc
ctgcgccatc 3300agatccttgg cggcaagaaa gccatccagt ttactttgca
gggcttccca accttaccag 3360agggcgcccc agctggcaat tccggttcgc
ttgctgtcca taaaaccgcc cagtctagca 3420actgttggga agggcgatcg
gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 3480gatgtgctgc
aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac
3534133534DNAartificial sequenceThe operatively linked components
of the TV-GHRH plasmid. 13gttgtaaaac gacggccagt gaattgtaat
acgactcact atagggcgaa ttggagctcc 60accgcggtgg cggccgtccg ccctcggcac
catcctcacg acacccaaat atggcgacgg 120gtgaggaatg gtggggagtt
atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180tggcgctcta
aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca
240aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg
gccggggccg 300cattcctggg ggccgggcgg tgctcccgcc cgcctcgata
aaaggctccg gggccggcgg 360cggcccacga gctacccgga ggagcgggag
gcgccaagct ctagaactag tggatcccaa 420ggcccaactc cccgaaccac
tcagggtcct gtggacagct cacctagctg ccatggtgct 480ctgggtgttc
ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc
540ccctttgacc ctcaggatgc ggcggtatgt agatgccatc ttcaccaaca
gctaccggaa 600ggtgctggcc cagctgtccg cccgcaagct gctccaggac
atcctgaaca ggcagcaggg 660agagaggaac caagagcaag gagcataatg
actgcaggaa ttcgatatca agcttatcgg 720ggtggcatcc ctgtgacccc
tccccagtgc ctctcctggc cctggaagtt gccactccag 780tgcccaccag
ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct
840tctataatat tatggggtgg aggggggtgg tatggagcaa ggggcaagtt
gggaagacaa 900cctgtagggc ctgcggggtc tattgggaac caagctggag
tgcagtggca caatcttggc 960tcactgcaat ctccgcctcc tgggttcaag
cgattctcct gcctcagcct cccgagttgt 1020tgggattcca ggcatgcatg
accaggctca gctaattttt gtttttttgg tagagacggg 1080gtttcaccat
attggccagg ctggtctcca actcctaatc tcaggtgatc tacccacctt
1140ggcctcccaa attgctggga ttacaggcgt gaaccactgc tcccttccct
gtccttctga 1200ttttaaaata actataccag caggaggacg tccagacaca
gcataggcta cctggccatg 1260cccaaccggt gggacatttg agttgcttgc
ttggcactgt cctctcatgc gttgggtcca 1320ctcagtagat gcctgttgaa
ttcgataccg tcgacctcga gggggggccc ggtaccagct 1380tttgttccct
ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc
1440ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
agcataaagt 1500gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
aattgcgttg cgctcactgc 1560ccgctttcca gtcgggaaac ctgtcgtgcc
agctgcatta atgaatcggc caacgcgcgg 1620ggagaggcgg tttgcgtatt
gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 1680cggtcgttcg
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
1740cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
aaggccagga 1800accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
ccgcccccct gacgagcatc 1860acaaaaatcg acgctcaagt cagaggtggc
gaaacccgac aggactataa agataccagg 1920cgtttccccc tggaagctcc
ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 1980acctgtccgc
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
2040atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
ccccccgttc 2100agcccgaccg ctgcgcctta tccggtaact atcgtcttga
gtccaacccg gtaagacacg 2160acttatcgcc actggcagca gccactggta
acaggattag cagagcgagg tatgtaggcg 2220gtgctacaga gttcttgaag
tggtggccta actacggcta cactagaaga acagtatttg 2280gtatctgcgc
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
2340gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
attacgcgca 2400gaaaaaaagg atctcaagaa gatcctttga tcttttctac
ggggtctgac gctcagaaga 2460actcgtcaag aaggcgatag aaggcgatgc
gctgcgaatc gggagcggcg ataccgtaaa 2520gcacgaggaa gcggtcagcc
cattcgccgc caagctcttc agcaatatca cgggtagcca 2580acgctatgtc
ctgatagcgg tccgccacac ccagccggcc acagtcgatg aatccagaaa
2640agcggccatt ttccaccatg atattcggca agcaggcatc gccatgggtc
acgacgagat 2700cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag
ttcggctggc gcgagcccct 2760gatgctcttc gtccagatca tcctgatcga
caagaccggc ttccatccga gtacgtgctc 2820gctcgatgcg atgtttcgct
tggtggtcga atgggcaggt agccggatca agcgtatgca 2880gccgccgcat
tgcatcagcc atgatggata ctttctcggc aggagcaagg tgagatgaca
2940ggagatcctg ccccggcact tcgcccaata gcagccagtc ccttcccgct
tcagtgacaa 3000cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag
ccacgatagc cgcgctgcct 3060cgtcctgcag ttcattcagg gcaccggaca
ggtcggtctt gacaaaaaga accgggcgcc 3120cctgcgctga cagccggaac
acggcggcat cagagcagcc gattgtctgt tgtgcccagt 3180catagccgaa
tagcctctcc acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt
3240caatcatgcg aaacgatcct catcctgtct cttgatcaga tcttgatccc
ctgcgccatc 3300agatccttgg cggcaagaaa gccatccagt ttactttgca
gggcttccca accttaccag 3360agggcgcccc agctggcaat tccggttcgc
ttgctgtcca taaaaccgcc cagtctagca 3420actgttggga agggcgatcg
gtgcgggcct cttcgctatt acgccagctg gcgaaagggg 3480gatgtgctgc
aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgac
3534143534DNAartificial sequenceThe operatively linked components
of the 15/27/28 GHRH plasmid. 14gttgtaaaac gacggccagt gaattgtaat
acgactcact atagggcgaa ttggagctcc 60accgcggtgg cggccgtccg ccctcggcac
catcctcacg acacccaaat atggcgacgg 120gtgaggaatg gtggggagtt
atttttagag cggtgaggaa ggtgggcagg cagcaggtgt 180tggcgctcta
aaaataactc ccgggagtta tttttagagc ggaggaatgg tggacaccca
240aatatggcga cggttcctca cccgtcgcca tatttgggtg tccgccctcg
gccggggccg 300cattcctggg ggccgggcgg tgctcccgcc cgcctcgata
aaaggctccg gggccggcgg 360cggcccacga gctacccgga ggagcgggag
gcgccaagct ctagaactag tggatcccaa 420ggcccaactc cccgaaccac
tcagggtcct gtggacagct cacctagctg ccatggtgct 480ctgggtgttc
ttctttgtga tcctcaccct cagcaacagc tcccactgct ccccacctcc
540ccctttgacc ctcaggatgc ggcggtatat cgatgccatc ttcaccaaca
gctaccggaa 600ggtgctggcc cagctgtccg cccgcaagct gctccaggac
atcctgaaca ggcagcaggg 660agagaggaac caagagcaag gagcataatg
actgcaggaa ttcgatatca agcttatcgg 720ggtggcatcc ctgtgacccc
tccccagtgc ctctcctggc cctggaagtt gccactccag 780tgcccaccag
ccttgtccta ataaaattaa gttgcatcat tttgtctgac taggtgtcct
840tctataatat
tatggggtgg aggggggtgg tatggagcaa ggggcaagtt gggaagacaa
900cctgtagggc ctgcggggtc tattgggaac caagctggag tgcagtggca
caatcttggc 960tcactgcaat ctccgcctcc tgggttcaag cgattctcct
gcctcagcct cccgagttgt 1020tgggattcca ggcatgcatg accaggctca
gctaattttt gtttttttgg tagagacggg 1080gtttcaccat attggccagg
ctggtctcca actcctaatc tcaggtgatc tacccacctt 1140ggcctcccaa
attgctggga ttacaggcgt gaaccactgc tcccttccct gtccttctga
1200ttttaaaata actataccag caggaggacg tccagacaca gcataggcta
cctggccatg 1260cccaaccggt gggacatttg agttgcttgc ttggcactgt
cctctcatgc gttgggtcca 1320ctcagtagat gcctgttgaa ttcgataccg
tcgacctcga gggggggccc ggtaccagct 1380tttgttccct ttagtgaggg
ttaatttcga gcttggcgta atcatggtca tagctgtttc 1440ctgtgtgaaa
ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
1500gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
cgctcactgc 1560ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
atgaatcggc caacgcgcgg 1620ggagaggcgg tttgcgtatt gggcgctctt
ccgcttcctc gctcactgac tcgctgcgct 1680cggtcgttcg gctgcggcga
gcggtatcag ctcactcaaa ggcggtaata cggttatcca 1740cagaatcagg
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
1800accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
gacgagcatc 1860acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
aggactataa agataccagg 1920cgtttccccc tggaagctcc ctcgtgcgct
ctcctgttcc gaccctgccg cttaccggat 1980acctgtccgc ctttctccct
tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 2040atctcagttc
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
2100agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
gtaagacacg 2160acttatcgcc actggcagca gccactggta acaggattag
cagagcgagg tatgtaggcg 2220gtgctacaga gttcttgaag tggtggccta
actacggcta cactagaaga acagtatttg 2280gtatctgcgc tctgctgaag
ccagttacct tcggaaaaag agttggtagc tcttgatccg 2340gcaaacaaac
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
2400gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
gctcagaaga 2460actcgtcaag aaggcgatag aaggcgatgc gctgcgaatc
gggagcggcg ataccgtaaa 2520gcacgaggaa gcggtcagcc cattcgccgc
caagctcttc agcaatatca cgggtagcca 2580acgctatgtc ctgatagcgg
tccgccacac ccagccggcc acagtcgatg aatccagaaa 2640agcggccatt
ttccaccatg atattcggca agcaggcatc gccatgggtc acgacgagat
2700cctcgccgtc gggcatgcgc gccttgagcc tggcgaacag ttcggctggc
gcgagcccct 2760gatgctcttc gtccagatca tcctgatcga caagaccggc
ttccatccga gtacgtgctc 2820gctcgatgcg atgtttcgct tggtggtcga
atgggcaggt agccggatca agcgtatgca 2880gccgccgcat tgcatcagcc
atgatggata ctttctcggc aggagcaagg tgagatgaca 2940ggagatcctg
ccccggcact tcgcccaata gcagccagtc ccttcccgct tcagtgacaa
3000cgtcgagcac agctgcgcaa ggaacgcccg tcgtggccag ccacgatagc
cgcgctgcct 3060cgtcctgcag ttcattcagg gcaccggaca ggtcggtctt
gacaaaaaga accgggcgcc 3120cctgcgctga cagccggaac acggcggcat
cagagcagcc gattgtctgt tgtgcccagt 3180catagccgaa tagcctctcc
acccaagcgg ccggagaacc tgcgtgcaat ccatcttgtt 3240caatcatgcg
aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc
3300agatccttgg cggcaagaaa gccatccagt ttactttgca gggcttccca
accttaccag 3360agggcgcccc agctggcaat tccggttcgc ttgctgtcca
taaaaccgcc cagtctagca 3420actgttggga agggcgatcg gtgcgggcct
cttcgctatt acgccagctg gcgaaagggg 3480gatgtgctgc aaggcgatta
agttgggtaa cgccagggtt ttcccagtca cgac 3534153534DNAartificial
sequenceThis is the entire plasmid sequence for wildtype GHRH.
15gttgtaaaac gacggccagt gaattgtaat acgactcact atagggcgaa ttggagctcc
60accgcggtgg cggccgtccg ccctcggcac catcctcacg acacccaaat atggcgacgg
120gtgaggaatg gtggggagtt atttttagag cggtgaggaa ggtgggcagg
cagcaggtgt 180tggcgctcta aaaataactc ccgggagtta tttttagagc
ggaggaatgg tggacaccca 240aatatggcga cggttcctca cccgtcgcca
tatttgggtg tccgccctcg gccggggccg 300cattcctggg ggccgggcgg
tgctcccgcc cgcctcgata aaaggctccg gggccggcgg 360cggcccacga
gctacccgga ggagcgggag gcgccaagct ctagaactag tggatcccaa
420ggcccaactc cccgaaccac tcagggtcct gtggacagct cacctagctg
ccatggtgct 480ctgggtgttc ttctttgtga tcctcaccct cagcaacagc
tcccactgct ccccacctcc 540ccctttgacc ctcaggatgc ggcggtatgc
agatgccatc ttcaccaaca gctaccggaa 600ggtgctgggc cagctgtccg
cccgcaagct gctccaggac atcatgagca ggcagcaggg 660agagaggaac
caagagcaag gagcataatg actgcaggaa ttcgatatca agcttatcgg
720ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
gccactccag 780tgcccaccag ccttgtccta ataaaattaa gttgcatcat
tttgtctgac taggtgtcct 840tctataatat tatggggtgg aggggggtgg
tatggagcaa ggggcaagtt gggaagacaa 900cctgtagggc ctgcggggtc
tattgggaac caagctggag tgcagtggca caatcttggc 960tcactgcaat
ctccgcctcc tgggttcaag cgattctcct gcctcagcct cccgagttgt
1020tgggattcca ggcatgcatg accaggctca gctaattttt gtttttttgg
tagagacggg 1080gtttcaccat attggccagg ctggtctcca actcctaatc
tcaggtgatc tacccacctt 1140ggcctcccaa attgctggga ttacaggcgt
gaaccactgc tcccttccct gtccttctga 1200ttttaaaata actataccag
caggaggacg tccagacaca gcataggcta cctggccatg 1260cccaaccggt
gggacatttg agttgcttgc ttggcactgt cctctcatgc gttgggtcca
1320ctcagtagat gcctgttgaa ttcgataccg tcgacctcga gggggggccc
ggtaccagct 1380tttgttccct ttagtgaggg ttaatttcga gcttggcgta
atcatggtca tagctgtttc 1440ctgtgtgaaa ttgttatccg ctcacaattc
cacacaacat acgagccgga agcataaagt 1500gtaaagcctg gggtgcctaa
tgagtgagct aactcacatt aattgcgttg cgctcactgc 1560ccgctttcca
gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
1620ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
tcgctgcgct 1680cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
ggcggtaata cggttatcca 1740cagaatcagg ggataacgca ggaaagaaca
tgtgagcaaa aggccagcaa aaggccagga 1800accgtaaaaa ggccgcgttg
ctggcgtttt tccataggct ccgcccccct gacgagcatc 1860acaaaaatcg
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
1920cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
cttaccggat 1980acctgtccgc ctttctccct tcgggaagcg tggcgctttc
tcatagctca cgctgtaggt 2040atctcagttc ggtgtaggtc gttcgctcca
agctgggctg tgtgcacgaa ccccccgttc 2100agcccgaccg ctgcgcctta
tccggtaact atcgtcttga gtccaacccg gtaagacacg 2160acttatcgcc
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
2220gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
acagtatttg 2280gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
agttggtagc tcttgatccg 2340gcaaacaaac caccgctggt agcggtggtt
tttttgtttg caagcagcag attacgcgca 2400gaaaaaaagg atctcaagaa
gatcctttga tcttttctac ggggtctgac gctcagaaga 2460actcgtcaag
aaggcgatag aaggcgatgc gctgcgaatc gggagcggcg ataccgtaaa
2520gcacgaggaa gcggtcagcc cattcgccgc caagctcttc agcaatatca
cgggtagcca 2580acgctatgtc ctgatagcgg tccgccacac ccagccggcc
acagtcgatg aatccagaaa 2640agcggccatt ttccaccatg atattcggca
agcaggcatc gccatgggtc acgacgagat 2700cctcgccgtc gggcatgcgc
gccttgagcc tggcgaacag ttcggctggc gcgagcccct 2760gatgctcttc
gtccagatca tcctgatcga caagaccggc ttccatccga gtacgtgctc
2820gctcgatgcg atgtttcgct tggtggtcga atgggcaggt agccggatca
agcgtatgca 2880gccgccgcat tgcatcagcc atgatggata ctttctcggc
aggagcaagg tgagatgaca 2940ggagatcctg ccccggcact tcgcccaata
gcagccagtc ccttcccgct tcagtgacaa 3000cgtcgagcac agctgcgcaa
ggaacgcccg tcgtggccag ccacgatagc cgcgctgcct 3060cgtcctgcag
ttcattcagg gcaccggaca ggtcggtctt gacaaaaaga accgggcgcc
3120cctgcgctga cagccggaac acggcggcat cagagcagcc gattgtctgt
tgtgcccagt 3180catagccgaa tagcctctcc acccaagcgg ccggagaacc
tgcgtgcaat ccatcttgtt 3240caatcatgcg aaacgatcct catcctgtct
cttgatcaga tcttgatccc ctgcgccatc 3300agatccttgg cggcaagaaa
gccatccagt ttactttgca gggcttccca accttaccag 3360agggcgcccc
agctggcaat tccggttcgc ttgctgtcca taaaaccgcc cagtctagca
3420actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
gcgaaagggg 3480gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
ttcccagtca cgac 3534164260DNAArtificial sequenceThis is the
sequence for the pSP-SEAP cDNA construct 16ggccgtccgc cttcggcacc
atcctcacga cacccaaata tggcgacggg tgaggaatgg 60tggggagtta tttttagagc
ggtgaggaag gtgggcaggc agcaggtgtt ggcgctctaa 120aaataactcc
cgggagttat ttttagagcg gaggaatggt ggacacccaa atatggcgac
180ggttcctcac ccgtcgccat atttgggtgt ccgccctcgg ccggggccgc
attcctgggg 240gccgggcggt gctcccgccc gcctcgataa aaggctccgg
ggccggcggc ggcccacgag 300ctacccggag gagcgggagg cgccaagctc
tagaactagt ggatcccccg ggctgcagga 360attcgatatc aagcttcgaa
tcgcgaattc gcccaccatg ctgctgctgc tgctgctgct 420gggcctgagg
ctacagctct ccctgggcat catcccagtt gaggaggaga acccggactt
480ctggaaccgc gaggcagccg aggccctggg tgccgccaag aagctgcagc
ctgcacagac 540agccgccaag aacctcatca tcttcctggg cgatgggatg
ggggtgtcta cggtgacagc 600tgccaggatc ctaaaagggc agaagaagga
caaactgggg cctgagatac ccctggccat 660ggaccgcttc ccatatgtgg
ctctgtccaa gacatacaat gtagacaaac atgtgccaga 720cagtggagcc
acagccacgg cctacctgtg cggggtcaag ggcaacttcc agaccattgg
780cttgagtgca gccgcccgct ttaaccagtg caacacgaca cgcggcaacg
aggtcatctc 840cgtgatgaat cgggccaaga aagcagggaa gtcagtggga
gtggtaacca ccacacgagt 900gcagcacgcc tcgccagccg gcacctacgc
ccacacggtg aaccgcaact ggtactcgga 960cgccgacgtg cctgcctcgg
cccgccagga ggggtgccag gacatcgcta cgcagctcat 1020ctccaacatg
gacattgacg tgatcctagg tggaggccga aagtacatgt ttcgcatggg
1080aaccccagac cctgagtacc cagatgacta cagccaaggt gggaccaggc
tggacgggaa 1140gaatctggtg caggaatggc tggcgaagcg ccagggtgcc
cggtatgtgt ggaaccgcac 1200tgagctcatg caggcttccc tggacccgtc
tgtgacccat ctcatgggtc tctttgagcc 1260tggagacatg aaatacgaga
tccaccgaga ctccacactg gacccctccc tgatggagat 1320gacagaggct
gccctgcgcc tgctgagcag gaacccccgc ggcttcttcc tcttcgtgga
1380gggtggtcgc atcgaccatg gtcatcatga aagcagggct taccgggcac
tgactgagac 1440gatcatgttc gacgacgcca ttgagagggc gggccagctc
accagcgagg aggacacgct 1500gagcctcgtc actgccgacc actcccacgt
cttctccttc ggaggctacc ccctgcgagg 1560gagctccatc ttcgggctgg
cccctggcaa ggcccgggac aggaaggcct acacggtcct 1620cctatacgga
aacggtccag gctatgtgct caaggacggc gcccggccgg atgttaccga
1680gagcgagagc gggagccccg agtatcggca gcagtcagca gtgcccctgg
acgaagagac 1740ccacgcaggc gaggacgtgg cggtgttcgc gcgcggcccg
caggcgcacc tggttcacgg 1800cgtgcaggag cagaccttca tagcgcacgt
catggccttc gccgcctgcc tggagcccta 1860caccgcctgc gacctggcgc
cccccgccgg caccaccgac gccgcgcacc cgggttactc 1920tagagtcggg
gcggccggcc gcttcgagca gacatgataa gatacattga tgagtttgga
1980caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
tgatgctatt 2040gctttatttg taaccattat aagctgcaat aaacaagtta
acaacaacaa ttgcattcat 2100tttatgtttc aggttcaggg ggaggtgtgg
gaggtttttt aaagcaagta aaacctctac 2160aaatgtggta aaatcgataa
ggatccgtcg accgatgccc ttgagagcct tcaacccagt 2220cagctccttc
cggtgggcgc ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt
2280tatcatgcaa ctcgtaggac aggtgccggc agcgctcttc cgcttcctcg
ctcactgact 2340cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
tcactcaaag gcggtaatac 2400ggttatccac agaatcaggg gataacgcag
gaaagaacat gtgagcaaaa ggccagcaaa 2460aggccaggaa ccgtaaaaag
gccgcgttgc tggcgttttt ccataggctc cgcccccctg 2520acgagcatca
caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
2580gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
accctgccgc 2640ttaccggata cctgtccgcc tttctccctt cgggaagcgt
ggcgctttct catagctcac 2700gctgtaggta tctcagttcg gtgtaggtcg
ttcgctccaa gctgggctgt gtgcacgaac 2760cccccgttca gcccgaccgc
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 2820taagacacga
cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
2880atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
actagaagga 2940cagtatttgg tatctgcgct ctgctgaagc cagttacctt
cggaaaaaga gttggtagct 3000cttgatccgg caaacaaacc accgctggta
gcggtggttt ttttgtttgc aagcagcaga 3060ttacgcgcag aaaaaaagga
tctcaagaag atcctttgat cttttctacg gggtctgacg 3120ctcagtggaa
cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
3180tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
atatatgagt 3240aaacttggtc tgacagttac caatgcttaa tcagtgaggc
acctatctca gcgatctgtc 3300tatttcgttc atccatagtt gcctgactcc
ccgtcgtgta gataactacg atacgggagg 3360gcttaccatc tggccccagt
gctgcaatga taccgcgaga cccacgctca ccggctccag 3420atttatcagc
aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
3480tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
agttcgccag 3540ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
cgtggtgtca cgctcgtcgt 3600ttggtatggc ttcattcagc tccggttccc
aacgatcaag gcgagttaca tgatccccca 3660tgttgtgcaa aaaagcggtt
agctccttcg gtcctccgat cgttgtcaga agtaagttgg 3720ccgcagtgtt
atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
3780ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
gaatagtgta 3840tgcggcgacc gagttgctct tgcccggcgt caatacggga
taataccgcg ccacatagca 3900gaactttaaa agtgctcatc attggaaaac
gttcttcggg gcgaaaactc tcaaggatct 3960taccgctgtt gagatccagt
tcgatgtaac ccactcgtgc acccaactga tcttcagcat 4020cttttacttt
caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
4080agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
caatattatt 4140gaagcattta tcagggttat tgtctcatga gcggatacat
atttgaatgt atttagaaaa 4200ataaacaaat aggggttccg cgcacatttc
cccgaaaagt gccacctgac gcgccctgta 4260172710DNAartificial
sequencePlasmid vector having GHRH optimized for mouse.
17tgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc cgtccgccct
60cggcaccatc ctcacgacac ccaaatatgg cgacgggtga ggaatggtgg ggagttattt
120ttagagcggt gaggaaggtg ggcaggcagc aggtgttggc gctctaaaaa
taactcccgg 180gagttatttt tagagcggag gaatggtgga cacccaaata
tggcgacggt tcctcacccg 240tcgccatatt tgggtgtccg ccctcggccg
gggccgcatt cctgggggcc gggcggtgct 300cccgcccgcc tcgataaaag
gctccggggc cggcggcggc ccacgagcta cccggaggag 360cgggaggcgc
caagcggatc ccaaggccca actccccgaa ccactcaggg tcctgtggac
420agctcaccta gctgccatgg tgctctgggt gctctttgtg atcctcatcc
tcaccagcgg 480cagccactgc agcctgcctc ccagccctcc cttcaggatg
cagaggcacg tggacgccat 540cttcaccacc aactacagga agctgctgag
ccagctgtac gccaggaagg tgatccagga 600catcatgaac aagcagggcg
agaggatcca ggagcagagg gccaggctga gctgataagc 660ttatcggggt
ggcatccctg tgacccctcc ccagtgcctc tcctggccct ggaagttgcc
720actccagtgc ccaccagcct tgtcctaata aaattaagtt gcatcatttt
gtctgactag 780gtgtccttct ataatattat ggggtggagg ggggtggtat
ggagcaaggg gcaagttggg 840aagacaacct gtagggctcg agggggggcc
cggtaccagc ttttgttccc tttagtgagg 900gttaatttcg agcttggtct
tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc 960ggctgcggcg
agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
1020gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa 1080aggccgcgtt gctggcgttt ttccataggc tccgcccccc
tgacgagcat cacaaaaatc 1140gacgctcaag tcagaggtgg cgaaacccga
caggactata aagataccag gcgtttcccc 1200ctggaagctc cctcgtgcgc
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg 1260cctttctccc
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
1320cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
cagcccgacc 1380gctgcgcctt atccggtaac tatcgtcttg agtccaaccc
ggtaagacac gacttatcgc 1440cactggcagc agccactggt aacaggatta
gcagagcgag gtatgtaggc ggtgctacag 1500agttcttgaa gtggtggcct
aactacggct acactagaag aacagtattt ggtatctgcg 1560ctctgctgaa
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
1620ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
agaaaaaaag 1680gatctcaaga agatcctttg atcttttcta cggggctagc
gcttagaaga actcatccag 1740cagacggtag aatgcaatac gttgagagtc
tggagctgca ataccataca gaaccaggaa 1800acggtcagcc cattcaccac
ccagttcctc tgcaatgtca cgggtagcca gtgcaatgtc 1860ctggtaacgg
tctgcaacac ccagacgacc acagtcaatg aaaccagaga aacgaccatt
1920ctcaaccatg atgttcggca ggcatgcatc accatgagta actaccaggt
cctcaccatc 1980cggcatacga gctttcagac gtgcaaacag ttcagccggt
gccagaccct gatgttcctc 2040atccaggtca tcctggtcaa ccagacctgc
ttccatacgg gtacgagcac gttcaatacg 2100atgttttgcc tggtggtcaa
acggacaggt agctgggtcc agggtgtgca gacgacgcat 2160tgcatcagcc
atgatagaaa ctttctctgc cggagccagg tgagaagaca gcaggtcctg
2220acccggaact tcacccagca gcagccagtc acgaccagct tcagtaacta
catccagaac 2280tgcagcacac ggaacaccag tggttgccag ccaagacaga
cgagctgctt catcctgcag 2340ttcattcaga gcaccagaca ggtcagtttt
aacaaacaga actggacgac cctgtgcaga 2400cagacggaaa acagctgcat
cagagcaacc aatggtctgc tgtgcccagt cataaccaaa 2460cagacgttca
acccaggctg ccggagaacc tgcatgcaga ccatcctgtt caatcatgcg
2520aaacgatcct catcctgtct cttgatcaga tcttgatccc ctgcgccatc
agatccttgg 2580cggcaagaaa gccatccagt ttactttgca gggcttccca
accttaccag agggcgcccc 2640agctggcaat tccggttcgc ttgctgtcca
taaaaccgcc cagtctagca actgttggga 2700agggcgatcg
2710182713DNAartificial sequencePlasmid vector having GHRH
optimized for rat. 18tgtaatacga ctcactatag ggcgaattgg agctccaccg
cggtggcggc cgtccgccct 60cggcaccatc ctcacgacac ccaaatatgg cgacgggtga
ggaatggtgg ggagttattt 120ttagagcggt gaggaaggtg ggcaggcagc
aggtgttggc gctctaaaaa taactcccgg 180gagttatttt tagagcggag
gaatggtgga cacccaaata tggcgacggt tcctcacccg 240tcgccatatt
tgggtgtccg ccctcggccg gggccgcatt cctgggggcc gggcggtgct
300cccgcccgcc tcgataaaag gctccggggc cggcggcggc ccacgagcta
cccggaggag 360cgggaggcgc caagcggatc ccaaggccca actccccgaa
ccactcaggg tcctgtggac 420agctcaccta gctgccatgg ccctgtgggt
gttcttcgtg ctgctgaccc tgaccagcgg 480aagccactgc agcctgcctc
ccagccctcc cttcagggtg cgccggcacg ccgacgccat 540cttcaccagc
agctacagga ggatcctggg ccagctgtac gctaggaagc tcctgcacga
600gatcatgaac aggcagcagg gcgagaggaa ccaggagcag aggagcaggt
tcaactgata 660agcttatcgg ggtggcatcc ctgtgacccc tccccagtgc
ctctcctggc cctggaagtt 720gccactccag tgcccaccag ccttgtccta
ataaaattaa gttgcatcat tttgtctgac 780taggtgtcct tctataatat
tatggggtgg aggggggtgg tatggagcaa ggggcaagtt 840gggaagacaa
cctgtagggc tcgagggggg gcccggtacc agcttttgtt ccctttagtg
900agggttaatt tcgagcttgg tcttccgctt cctcgctcac tgactcgctg
cgctcggtcg 960ttcggctgcg gcgagcggta tcagctcact caaaggcggt
aatacggtta tccacagaat 1020caggggataa cgcaggaaag aacatgtgag
caaaaggcca gcaaaaggcc aggaaccgta 1080aaaaggccgc gttgctggcg
tttttccata ggctccgccc ccctgacgag catcacaaaa 1140atcgacgctc
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
1200cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
ggatacctgt 1260ccgcctttct cccttcggga agcgtggcgc tttctcatag
ctcacgctgt aggtatctca 1320gttcggtgta ggtcgttcgc tccaagctgg
gctgtgtgca cgaacccccc gttcagcccg 1380accgctgcgc cttatccggt
aactatcgtc ttgagtccaa cccggtaaga cacgacttat 1440cgccactggc
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
1500cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
tttggtatct 1560gcgctctgct gaagccagtt accttcggaa aaagagttgg
tagctcttga tccggcaaac 1620aaaccaccgc tggtagcggt ggtttttttg
tttgcaagca gcagattacg cgcagaaaaa 1680aaggatctca agaagatcct
ttgatctttt ctacggggct agcgcttaga agaactcatc 1740cagcagacgg
tagaatgcaa tacgttgaga gtctggagct gcaataccat acagaaccag
1800gaaacggtca gcccattcac cacccagttc ctctgcaatg tcacgggtag
ccagtgcaat 1860gtcctggtaa cggtctgcaa cacccagacg accacagtca
atgaaaccag agaaacgacc 1920attctcaacc atgatgttcg gcaggcatgc
atcaccatga gtaactacca ggtcctcacc 1980atccggcata cgagctttca
gacgtgcaaa cagttcagcc ggtgccagac cctgatgttc 2040ctcatccagg
tcatcctggt caaccagacc tgcttccata cgggtacgag cacgttcaat
2100acgatgtttt gcctggtggt caaacggaca ggtagctggg tccagggtgt
gcagacgacg 2160cattgcatca gccatgatag aaactttctc tgccggagcc
aggtgagaag acagcaggtc 2220ctgacccgga acttcaccca gcagcagcca
gtcacgacca gcttcagtaa ctacatccag 2280aactgcagca cacggaacac
cagtggttgc cagccaagac agacgagctg cttcatcctg 2340cagttcattc
agagcaccag acaggtcagt tttaacaaac agaactggac gaccctgtgc
2400agacagacgg aaaacagctg catcagagca accaatggtc tgctgtgccc
agtcataacc 2460aaacagacgt tcaacccagg ctgccggaga acctgcatgc
agaccatcct gttcaatcat 2520gcgaaacgat cctcatcctg tctcttgatc
agatcttgat cccctgcgcc atcagatcct 2580tggcggcaag aaagccatcc
agtttacttt gcagggcttc ccaaccttac cagagggcgc 2640cccagctggc
aattccggtt cgcttgctgt ccataaaacc gcccagtcta gcaactgttg
2700ggaagggcga tcg 2713192704DNAartificial sequencePlasmid vector
having GHRH optimized for bovine. 19tgtaatacga ctcactatag
ggcgaattgg agctccaccg cggtggcggc cgtccgccct 60cggcaccatc ctcacgacac
ccaaatatgg cgacgggtga ggaatggtgg ggagttattt 120ttagagcggt
gaggaaggtg ggcaggcagc aggtgttggc gctctaaaaa taactcccgg
180gagttatttt tagagcggag gaatggtgga cacccaaata tggcgacggt
tcctcacccg 240tcgccatatt tgggtgtccg ccctcggccg gggccgcatt
cctgggggcc gggcggtgct 300cccgcccgcc tcgataaaag gctccggggc
cggcggcggc ccacgagcta cccggaggag 360cgggaggcgc caagcggatc
ccaaggccca actccccgaa ccactcaggg tcctgtggac 420agctcaccta
gctgccatgg tgctgtgggt gttcttcctg gtgaccctga ccctgagcag
480cggctcccac ggctccctgc cctcccagcc tctgcgcatc cctcgctacg
ccgacgccat 540cttcaccaac agctaccgca aggtgctcgg ccagctcagc
gcccgcaagc tcctgcagga 600catcatgaac cggcagcagg gcgagcgcaa
ccaggagcag ggagcctgat aagcttatcg 660gggtggcatc cctgtgaccc
ctccccagtg cctctcctgg ccctggaagt tgccactcca 720gtgcccacca
gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc
780ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt
tgggaagaca 840acctgtaggg ctcgaggggg ggcccggtac cagcttttgt
tccctttagt gagggttaat 900ttcgagcttg gtcttccgct tcctcgctca
ctgactcgct gcgctcggtc gttcggctgc 960ggcgagcggt atcagctcac
tcaaaggcgg taatacggtt atccacagaa tcaggggata 1020acgcaggaaa
gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
1080cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
aatcgacgct 1140caagtcagag gtggcgaaac ccgacaggac tataaagata
ccaggcgttt ccccctggaa 1200gctccctcgt gcgctctcct gttccgaccc
tgccgcttac cggatacctg tccgcctttc 1260tcccttcggg aagcgtggcg
ctttctcata gctcacgctg taggtatctc agttcggtgt 1320aggtcgttcg
ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
1380ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
tcgccactgg 1440cagcagccac tggtaacagg attagcagag cgaggtatgt
aggcggtgct acagagttct 1500tgaagtggtg gcctaactac ggctacacta
gaagaacagt atttggtatc tgcgctctgc 1560tgaagccagt taccttcgga
aaaagagttg gtagctcttg atccggcaaa caaaccaccg 1620ctggtagcgg
tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
1680aagaagatcc tttgatcttt tctacggggc tagcgcttag aagaactcat
ccagcagacg 1740gtagaatgca atacgttgag agtctggagc tgcaatacca
tacagaacca ggaaacggtc 1800agcccattca ccacccagtt cctctgcaat
gtcacgggta gccagtgcaa tgtcctggta 1860acggtctgca acacccagac
gaccacagtc aatgaaacca gagaaacgac cattctcaac 1920catgatgttc
ggcaggcatg catcaccatg agtaactacc aggtcctcac catccggcat
1980acgagctttc agacgtgcaa acagttcagc cggtgccaga ccctgatgtt
cctcatccag 2040gtcatcctgg tcaaccagac ctgcttccat acgggtacga
gcacgttcaa tacgatgttt 2100tgcctggtgg tcaaacggac aggtagctgg
gtccagggtg tgcagacgac gcattgcatc 2160agccatgata gaaactttct
ctgccggagc caggtgagaa gacagcaggt cctgacccgg 2220aacttcaccc
agcagcagcc agtcacgacc agcttcagta actacatcca gaactgcagc
2280acacggaaca ccagtggttg ccagccaaga cagacgagct gcttcatcct
gcagttcatt 2340cagagcacca gacaggtcag ttttaacaaa cagaactgga
cgaccctgtg cagacagacg 2400gaaaacagct gcatcagagc aaccaatggt
ctgctgtgcc cagtcataac caaacagacg 2460ttcaacccag gctgccggag
aacctgcatg cagaccatcc tgttcaatca tgcgaaacga 2520tcctcatcct
gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa
2580gaaagccatc cagtttactt tgcagggctt cccaacctta ccagagggcg
ccccagctgg 2640caattccggt tcgcttgctg tccataaaac cgcccagtct
agcaactgtt gggaagggcg 2700atcg 2704202704DNAartificial
sequencePlasmid vector having GHRH optimized for ovine.
20tgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc cgtccgccct
60cggcaccatc ctcacgacac ccaaatatgg cgacgggtga ggaatggtgg ggagttattt
120ttagagcggt gaggaaggtg ggcaggcagc aggtgttggc gctctaaaaa
taactcccgg 180gagttatttt tagagcggag gaatggtgga cacccaaata
tggcgacggt tcctcacccg 240tcgccatatt tgggtgtccg ccctcggccg
gggccgcatt cctgggggcc gggcggtgct 300cccgcccgcc tcgataaaag
gctccggggc cggcggcggc ccacgagcta cccggaggag 360cgggaggcgc
caagcggatc ccaaggccca actccccgaa ccactcaggg tcctgtggac
420agctcaccta gctgccatgg tgctgtgggt gttcttcctg gtgaccctga
ccctgagcag 480cggaagccac ggcagcctgc ccagccagcc cctgaggatc
cctaggtacg ccgacgccat 540cttcaccaac agctacagga agatcctggg
ccagctgagc gctaggaagc tcctgcagga 600catcatgaac aggcagcagg
gcgagaggaa ccaggagcag ggcgcctgat aagcttatcg 660gggtggcatc
cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca
720gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga
ctaggtgtcc 780ttctataata ttatggggtg gaggggggtg gtatggagca
aggggcaagt tgggaagaca 840acctgtaggg ctcgaggggg ggcccggtac
cagcttttgt tccctttagt gagggttaat 900ttcgagcttg gtcttccgct
tcctcgctca ctgactcgct gcgctcggtc gttcggctgc 960ggcgagcggt
atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
1020acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
aaaaaggccg 1080cgttgctggc gtttttccat aggctccgcc cccctgacga
gcatcacaaa aatcgacgct 1140caagtcagag gtggcgaaac ccgacaggac
tataaagata ccaggcgttt ccccctggaa 1200gctccctcgt gcgctctcct
gttccgaccc tgccgcttac cggatacctg tccgcctttc 1260tcccttcggg
aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
1320aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
gaccgctgcg 1380ccttatccgg taactatcgt cttgagtcca acccggtaag
acacgactta tcgccactgg 1440cagcagccac tggtaacagg attagcagag
cgaggtatgt aggcggtgct acagagttct 1500tgaagtggtg gcctaactac
ggctacacta gaagaacagt atttggtatc tgcgctctgc 1560tgaagccagt
taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
1620ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
aaaggatctc 1680aagaagatcc tttgatcttt tctacggggc tagcgcttag
aagaactcat ccagcagacg 1740gtagaatgca atacgttgag agtctggagc
tgcaatacca tacagaacca ggaaacggtc 1800agcccattca ccacccagtt
cctctgcaat gtcacgggta gccagtgcaa tgtcctggta 1860acggtctgca
acacccagac gaccacagtc aatgaaacca gagaaacgac cattctcaac
1920catgatgttc ggcaggcatg catcaccatg agtaactacc aggtcctcac
catccggcat 1980acgagctttc agacgtgcaa acagttcagc cggtgccaga
ccctgatgtt cctcatccag 2040gtcatcctgg tcaaccagac ctgcttccat
acgggtacga gcacgttcaa tacgatgttt 2100tgcctggtgg tcaaacggac
aggtagctgg gtccagggtg tgcagacgac gcattgcatc 2160agccatgata
gaaactttct ctgccggagc caggtgagaa gacagcaggt cctgacccgg
2220aacttcaccc agcagcagcc agtcacgacc agcttcagta actacatcca
gaactgcagc 2280acacggaaca ccagtggttg ccagccaaga cagacgagct
gcttcatcct gcagttcatt 2340cagagcacca gacaggtcag ttttaacaaa
cagaactgga cgaccctgtg cagacagacg 2400gaaaacagct gcatcagagc
aaccaatggt ctgctgtgcc cagtcataac caaacagacg 2460ttcaacccag
gctgccggag aacctgcatg cagaccatcc tgttcaatca tgcgaaacga
2520tcctcatcct gtctcttgat cagatcttga tcccctgcgc catcagatcc
ttggcggcaa 2580gaaagccatc cagtttactt tgcagggctt cccaacctta
ccagagggcg ccccagctgg 2640caattccggt tcgcttgctg tccataaaac
cgcccagtct agcaactgtt gggaagggcg 2700atcg 2704212713DNAartificial
sequencePlasmid vector having GHRH optimized for chicken.
21tgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc cgtccgccct
60cggcaccatc ctcacgacac ccaaatatgg cgacgggtga ggaatggtgg ggagttattt
120ttagagcggt gaggaaggtg ggcaggcagc aggtgttggc gctctaaaaa
taactcccgg 180gagttatttt tagagcggag gaatggtgga cacccaaata
tggcgacggt tcctcacccg 240tcgccatatt tgggtgtccg ccctcggccg
gggccgcatt cctgggggcc gggcggtgct 300cccgcccgcc tcgataaaag
gctccggggc cggcggcggc ccacgagcta cccggaggag 360cgggaggcgc
caagcggatc ccaaggccca actccccgaa ccactcaggg tcctgtggac
420agctcaccta gctgccatgg ccctgtgggt gttctttgtg ctgctgaccc
tgacctccgg 480aagccactgc agcctgccac ccagcccacc cttccgcgtc
aggcgccacg ccgacggcat 540cttcagcaag gcctaccgca agctcctggg
ccagctgagc gcacgcaact acctgcacag 600cctgatggcc aagcgcgtgg
gcagcggact gggagacgag gccgagcccc tgagctgata 660agcttatcgg
ggtggcatcc ctgtgacccc tccccagtgc ctctcctggc cctggaagtt
720gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat
tttgtctgac 780taggtgtcct tctataatat tatggggtgg aggggggtgg
tatggagcaa ggggcaagtt 840gggaagacaa cctgtagggc tcgagggggg
gcccggtacc agcttttgtt ccctttagtg 900agggttaatt tcgagcttgg
tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 960ttcggctgcg
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
1020caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
aggaaccgta 1080aaaaggccgc gttgctggcg tttttccata ggctccgccc
ccctgacgag catcacaaaa 1140atcgacgctc aagtcagagg tggcgaaacc
cgacaggact ataaagatac caggcgtttc 1200cccctggaag ctccctcgtg
cgctctcctg ttccgaccct gccgcttacc ggatacctgt 1260ccgcctttct
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
1320gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
gttcagcccg 1380accgctgcgc cttatccggt aactatcgtc ttgagtccaa
cccggtaaga cacgacttat 1440cgccactggc agcagccact ggtaacagga
ttagcagagc gaggtatgta ggcggtgcta 1500cagagttctt gaagtggtgg
cctaactacg gctacactag aagaacagta tttggtatct 1560gcgctctgct
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
1620aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
cgcagaaaaa 1680aaggatctca agaagatcct ttgatctttt ctacggggct
agcgcttaga agaactcatc 1740cagcagacgg tagaatgcaa tacgttgaga
gtctggagct gcaataccat acagaaccag 1800gaaacggtca gcccattcac
cacccagttc ctctgcaatg tcacgggtag ccagtgcaat 1860gtcctggtaa
cggtctgcaa cacccagacg accacagtca atgaaaccag agaaacgacc
1920attctcaacc atgatgttcg gcaggcatgc atcaccatga gtaactacca
ggtcctcacc 1980atccggcata cgagctttca gacgtgcaaa cagttcagcc
ggtgccagac cctgatgttc 2040ctcatccagg tcatcctggt caaccagacc
tgcttccata cgggtacgag cacgttcaat 2100acgatgtttt gcctggtggt
caaacggaca ggtagctggg tccagggtgt gcagacgacg 2160cattgcatca
gccatgatag aaactttctc tgccggagcc aggtgagaag acagcaggtc
2220ctgacccgga acttcaccca gcagcagcca gtcacgacca gcttcagtaa
ctacatccag 2280aactgcagca cacggaacac cagtggttgc cagccaagac
agacgagctg cttcatcctg 2340cagttcattc agagcaccag acaggtcagt
tttaacaaac agaactggac gaccctgtgc 2400agacagacgg aaaacagctg
catcagagca accaatggtc tgctgtgccc agtcataacc 2460aaacagacgt
tcaacccagg ctgccggaga acctgcatgc agaccatcct gttcaatcat
2520gcgaaacgat cctcatcctg tctcttgatc agatcttgat cccctgcgcc
atcagatcct 2580tggcggcaag aaagccatcc agtttacttt gcagggcttc
ccaaccttac cagagggcgc 2640cccagctggc aattccggtt cgcttgctgt
ccataaaacc gcccagtcta gcaactgttg 2700ggaagggcga tcg
27132255DNAartificial sequenceNucleic acid sequence of hGH" 5' UTR.
22caaggcccaa ctccccgaac cactcagggt cctgtggaca gctcacctag ctgcc
5523782DNAartificial sequenceSequence of a plasmid pUC-18 origin of
replicaition 23tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
ttcggctgcg gcgagcggta 60tcagctcact caaaggcggt aatacggtta tccacagaat
caggggataa cgcaggaaag 120aacatgtgag caaaaggcca gcaaaaggcc
aggaaccgta aaaaggccgc gttgctggcg 180tttttccata ggctccgccc
ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240tggcgaaacc
cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
300cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
cccttcggga 360agcgtggcgc tttctcatag ctcacgctgt aggtatctca
gttcggtgta ggtcgttcgc 420tccaagctgg gctgtgtgca cgaacccccc
gttcagcccg accgctgcgc cttatccggt 480aactatcgtc ttgagtccaa
cccggtaaga cacgacttat cgccactggc agcagccact 540ggtaacagga
ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
600cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
gaagccagtt 660accttcggaa aaagagttgg tagctcttga tccggcaaac
aaaccaccgc tggtagcggt 720ggtttttttg tttgcaagca gcagattacg
cgcagaaaaa aaggatctca agaagatcct 780tt 782245DNAartificial
sequenceThis is a NEO ribosomal binding site. 24tcctc
52529DNAartificial sequenceNucleic acid sequence of a prokaryotic
PNEO promoter. 25accttaccag agggcgcccc agctggcaa
29263558DNAartificial sequenceSequence for the inducible pGR1774
containing GHRH. 26atgcctggag acgccatcca cgctgttttg acctccatag
aagacaccgg gaccgatcca 60gcctccgcgg ccgggaacgg tgcattggaa cgcggattcc
ccgtgttaat taacaggtaa 120gtgtcttcct cctgtttcct tcccctgcta
ttctgctcaa ccttcctatc agaaactgca 180gtatctgtat ttttgctagc
agtaatacta acggttcttt ttttctcttc acaggccacc 240atgtagaact
agtgatccca aggcccaact ccccgaacca ctcagggtcc tgtggacagc
300tcacctagct gccatggtgc tctgggtgtt cttctttgtg atcctcaccc
tcagcaacag 360ctcccactgc tccccacctc cccctttgac cctcaggatg
cggcggtatg cagatgccat 420cttcaccaac agctaccgga aggtgctggg
ccagctgtcc gcccgcaagc tgctccagga 480catcatgagc aggcagcagg
gagagagcaa ccaagagcga ggagcataat gactgcagga 540attcgatatc
aagcttatcg gggtggcatc cctgtgaccc ctccccagtg cctctcctgg
600ccctggaagt tgccactcca gtgcccacca gccttgtcct aataaaatta
agttgcatca 660ttttgtctga ctaggtgtcc ttctataata ttatggggtg
gaggggggtg gtatggagca 720aggggcaagt tgggaagaca acctgtaggg
cctgcggggt ctattgggaa ccaagctgga 780gtgcagtggc acaatcttgg
ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc 840tgcctcagcc
tcccgagttg ttgggattcc aggcatgcat gaccaggctc agctaatttt
900tgtttttttg gtagagacgg ggtttcacca tattggccag gctggtctcc
aactcctaat 960ctcaggtgat ctacccacct tggcctccca aattgctggg
attacaggcg tgaaccactg 1020ctcccttccc tgtccttctg attttaaaat
aactatacca gcaggaggac gtccagacac 1080agcataggct acctggccat
gcccaaccgg tgggacattt gagttgcttg cttggcactg 1140tcctctcatg
cgttgggtcc actcagtaga tgcctgttga attcgatacc gtcgacctcg
1200agggggggcc cggtaccagc ttttgttccc tttagtgagg gttaatttcg
agcttggcgt 1260aatcatggtc atagctgttt cctgtgtgaa attgttatcc
gctcacaatt ccacacaaca 1320tacgagccgg aagcataaag tgtaaagcct
ggggtgccta atgagtgagc taactcacat 1380taattgcgtt gcgctcactg
cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 1440aatgaatcgg
ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
1500cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
gctcactcaa 1560aggcggtaat acggttatcc acagaatcag gggataacgc
aggaaagaac atgtgagcaa 1620aaggccagca aaaggccagg aaccgtaaaa
aggccgcgtt gctggcgttt ttccataggc 1680tccgcccccc tgacgagcat
cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 1740caggactata
aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
1800cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
gtggcgcttt 1860ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
cgttcgctcc aagctgggct 1920gtgtgcacga accccccgtt cagcccgacc
gctgcgcctt atccggtaac tatcgtcttg 1980agtccaaccc ggtaagacac
gacttatcgc cactggcagc agccactggt aacaggatta 2040gcagagcgag
gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
2100acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
ttcggaaaaa 2160gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
tagcggtggt ttttttgttt 2220gcaagcagca gattacgcgc agaaaaaaag
gatctcaaga agatcctttg atcttttcta 2280cggggtctga cgctcagaag
aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat 2340cgggagcggc
gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt
2400cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca
cccagccggc 2460cacagtcgat gaatccagaa aagcggccat tttccaccat
gatattcggc aagcaggcat 2520cgccatgggt cacgacgaga tcctcgccgt
cgggcatgcg cgccttgagc ctggcgaaca 2580gttcggctgg cgcgagcccc
tgatgctctt cgtccagatc atcctgatcg acaagaccgg 2640cttccatycg
agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg
2700tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat
actttctcgg 2760caggagcaag gtgagatgac aggagatcct gccccggcac
ttcgcccaat agcagccagt 2820cccttcccgc ttcagtgaca acgtcgagca
cagctgcgca aggaacgccc gtcgtggcca 2880gccacgatag ccgcgctgcc
tcgtcctgca gttcattcag ggcaccggac aggtcggtct 2940tgacaaaaag
aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc
3000cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg
gccggagaac 3060ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc
tcatcctgtc tcttgatcag 3120atcttgatcc cctgcgccat cagatccttg
gcggcaagaa agccatccag tttactttgc 3180agggcttccc aaccttacca
gagggcgccc cagctggcaa ttccggttcg cttgctgtcc 3240ataaaaccgc
ccagtctagc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
3300tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta
acgccagggt 3360tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
gtaatacgac tcactatagg 3420gcgaattaat tcgagcttgc atgcctgcag
ggtcgaagcg gagtactgtc ctccgagtgg 3480agtactgtcc tccgagcgga
gtactgtcct ccgagtcgag ggtcgaagcg gagtactgtc 3540ctccgagtgg agtactgt
3558274855DNAartificial SequenceSequence for the muscle-specific
GeneSwitch plasmid, pGS1633 27aggggccgct ctagctagag tctgcctgcc
ccctgcctgg cacagcccgt acctggccgc 60acgctccctc acaggtgaag ctcgaaaact
ccgtccccgt aaggagcccc gctgcccccc 120gaggcctcct ccctcacgcc
tcgctgcgct cccggctccc gcacggccct gggagaggcc 180cccaccgctt
cgtccttaac gggcccggcg gtgccggggg attatttcgg ccccggcccc
240gggggggccc
ggcagacgct ccttatacgg cccggcctcg ctcacctggg ccgcggccag
300gagcgccttc tttgggcagc gccgggccgg ggccgcgccg ggcccgacac
ccaaatatgg 360cgacggccgg ggccgcattc ctgggggccg ggcggtgctc
ccgcccgcct cgataaaagg 420ctccggggcc ggcgggcgac tcagatcgcc
tggagacgcc atccacgctg ttttgacctc 480catagaagac accgggaccg
atccagcctc cgcggccggg aacggtgcat tggaacgcgg 540attccccgtg
ttaattaaca ggtaagtgtc ttcctcctgt ttccttcccc tgctattctg
600ctcaaccttc ctatcagaaa ctgcagtatc tgtatttttg ctagcagtaa
tactaacggt 660tctttttttc tcttcacagg ccaccaagct accggtccac
catggactcc cagcagccag 720atctgaagct actgtcttct atcgaacaag
catgcgatat ttgccgactt aaaaagctca 780agtgctccaa agaaaaaccg
aagtgcgcca agtgtctgaa gaacaactgg gagtgtcgct 840actctcccaa
aaccaaaagg tctccgctga ctagggcaca tctgacagaa gtggaatcaa
900ggctagaaag actggaacag ctatttctac tgatttttcc tcgagaccag
aaaaagttca 960ataaagtcag agttgtgaga gcactggatg ctgttgctct
cccacagcca gtgggcgttc 1020caaatgaaag ccaagcccta agccagagat
tcactttttc accaggtcaa gacatacagt 1080tgattccacc actgatcaac
ctgttaatga gcattgaacc agatgtgatc tatgcaggac 1140atgacaacac
aaaacctgac acctccagtt ctttgctgac aagtcttaat caactaggcg
1200agaggcaact tctttcagta gtcaagtggt ctaaatcatt gccaggtttt
cgaaacttac 1260atattgatga ccagataact ctcattcagt attcttggat
gagcttaatg gtgtttggtc 1320taggatggag atcctacaaa cacgtcagtg
ggcagatgct gtattttgca cctgatctaa 1380tactaaatga acagcggatg
aaagaatcat cattctattc attatgcctt accatgtggc 1440agatcccaca
ggagtttgtc aagcttcaag ttagccaaga agagttcctc tgtatgaaag
1500tattgttact tcttaataca attcctttgg aagggctacg aagtcaaacc
cagtttgagg 1560agatgaggtc aagctacatt agagagctca tcaaggcaat
tggtttgagg caaaaaggag 1620ttgtgtcgag ctcacagcgt ttctatcaac
ttacaaaact tcttgataac ttgcatgatc 1680ttgtcaaaca acttcatctg
tactgcttga atacatttat ccagtcccgg gcactgagtg 1740ttgaatttcc
agaaatgatg tctgaagtta ttgctgggtc gacgcccatg gaattccagt
1800acctgccaga tacagacgat cgtcaccgga ttgaggagaa acgtaaaagg
acatatgaga 1860ccttcaagag catcatgaag aagagtcctt tcagcggacc
caccgacccc cggcctccac 1920ctcgacgcat tgctgtgcct tcccgcagct
cagcttctgt ccccaagcca gcaccccagc 1980cctatccctt tacgtcatcc
ctgagcacca tcaactatga tgagtttccc accatggtgt 2040ttccttctgg
gcagatcagc caggcctcgg ccttggcccc ggcccctccc caagtcctgc
2100cccaggctcc agcccctgcc cctgctccag ccatggtatc agctctggcc
caggccccag 2160cccctgtccc agtcctagcc ccaggccctc ctcaggctgt
ggccccacct gcccccaagc 2220ccacccaggc tggggaagga acgctgtcag
aggccctgct gcagctgcag tttgatgatg 2280aagacctggg ggccttgctt
ggcaacagca cagacccagc tgtgttcaca gacctggcat 2340ccgtcgacaa
ctccgagttt cagcagctgc tgaaccaggg catacctgtg gccccccaca
2400caactgagcc catgctgatg gagtaccctg aggctataac tcgcctagtg
acaggggccc 2460agaggccccc cgacccagct cctgctccac tgggggcccc
ggggctcccc aatggcctcc 2520tttcaggaga tgaagacttc tcctccattg
cggacatgga cttctcagcc ctgctgagtc 2580agatcagctc ctaaggatcc
tccggactag aaaagccgaa ttctgcagga attgggtggc 2640atccctgtga
cccctcccca gtgcctctcc tggccctgga agttgccact ccagtgccca
2700ccagccttgt cctaataaaa ttaagttgca tcattttgtc tgactaggtg
tccttctata 2760atattatggg gtggaggggg gtggtatgga gcaaggggca
agttgggaag acaacctgta 2820gggctcgagg gggggcccgg taccagcttt
tgttcccttt agtgagggtt aatttcgagc 2880ttggcgtaat catggtcata
gctgtttcct gtgtgaaatt gttatccgct cacaattcca 2940cacaacatac
gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
3000ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
gtcgtgccag 3060ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
tgcgtattgg gcgctcttcc 3120gcttcctcgc tcactgactc gctgcgctcg
gtcgttcggc tgcggcgagc ggtatcagct 3180cactcaaagg cggtaatacg
gttatccaca gaatcagggg ataacgcagg aaagaacatg 3240tgagcaaaag
gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
3300cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
gaggtggcga 3360aacccgacag gactataaag ataccaggcg tttccccctg
gaagctccct cgtgcgctct 3420cctgttccga ccctgccgct taccggatac
ctgtccgcct ttctcccttc gggaagcgtg 3480gcgctttctc atagctcacg
ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 3540ctgggctgtg
tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
3600cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
cactggtaac 3660aggattagca gagcgaggta tgtaggcggt gctacagagt
tcttgaagtg gtggcctaac 3720tacggctaca ctagaaggac agtatttggt
atctgcgctc tgctgaagcc agttaccttc 3780ggaaaaagag ttggtagctc
ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3840tttgtttgca
agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
3900ttttctacgg ggtctgacgc tcagaagaac tcgtcaagaa ggcgatagaa
ggcgatgcgc 3960tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc
ggtcagccca ttcgccgcca 4020agctcttcag caatatcacg ggtagccaac
gctatgtcct gatagcggtc cgccacaccc 4080agccggccac agtcgatgaa
tccagaaaag cggccatttt ccaccatgat attcggcaag 4140caggcatcgc
catgcgtcac gacgagatcc tcgccgtcgg gcatgcgcgc cttgagcctg
4200gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
ctgatcgaca 4260agaccggctt ccatccgagt acgtgctcgc tcgatgcgat
gtttcgcttg gtggtcgaat 4320gggcaggtag ccggatcaag cgtatgcagc
cgccgcattg catcagccat gatggatact 4380ttctcggcag gagcaaggtg
agatgacagg agatcctgcc ccggcacttc gcccaatagc 4440agccagtccc
ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc
4500gtggccagcc acgatagccg cgctgcctcg tcctgcagtt cattcagggc
accggacagg 4560tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca
gccggaacac ggcggcatca 4620gagcagccga ttgtctgttg tgcccagtca
tagccgaata gcctctccac ccaagcggcc 4680ggagaacctg cgtgcaatcc
atcttgttca atcatgcgaa acgatcctca tcctgtctct 4740tgatcagatc
ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt
4800actttgcagg gcttcccaac cttaccagag ggcgaattcg agcttgcatg cctgc
48552811DNAArtificial sequenceThis is the restriction cleavage site
for enzyme Ahd I 28gacnnnnngt c 11299DNAartificial sequenceThis is
the restriction cleavage site for AlwN I 29cagnnnctg
93013DNAartificial sequenceThis is the restriction cleavage site
for Bae I 30nacnnnngta ycn 133112DNAartificial sequenceThis is the
restriction cleavage site for Bcg I 31cgannnnnnt gc
123211DNAartificial sequenceThis is the restriction cleavage site
for Bgl I 32gccnnnnngg c 113310DNAartificial sequenceThis is the
restriction cleavage site for BsaB I 33gatnnnnatc
103411DNAartificial sequenceThis is the restriction cleavage site
for Bsl I. 34ccnnnnnnng g 113511DNAartificial sequenceThis is the
restriction cleavage site for BstAP I 35gcannnnntg c
113612DNAartificial sequenceThis is the restriction cleavage site
for BstX I 36ccannnnnnt gg 12379DNAartificial sequenceThis is the
restriction cleavage site for Dra III 37cacnnngtg
93812DNAartificial sequenceThis is the restriction cleavage site
for Drd I 38gacnnnnnng tc 123911DNAartificial sequenceThis is the
restriction cleavage site for EcoN I 39cctnnnnnag g
11409DNAartificial sequenceThis is the restriction cleavage site
for Fau I 40cccgcnnnn 94110DNAartificial sequenceThis is the
restriction cleavage site for Mly I 41gagtcnnnnn
104210DNAartificial sequenceThis is the restriction cleavage site
for Msl I. 42caynnnnrtg 104311DNAartificial sequenceThis is the
restriction cleavage site for Mwo I. 43gcnnnnnnng c
11449DNAArtificial SequenceThis is the restriction cleavage site
for PflF I 44gacnnngtc 94511DNAArtificial SequenceThis is the
restriction cleavage site for PflM I. 45ccannnnntg g
114610DNAartificial sequenceThis is the restriction cleavage site
for PshA I. 46gacnnnngtc 104713DNAartificial sequenceThis is the
restriction cleavage site for Sfi I 47ggccnnnnng gcc
13489DNAartificial sequenceThis is the restriction cleavage site
for Tth1 111I 48gacnnngtc 94915DNAartificial sequenceThis is the
restriction cleavage site for Xcm I 49ccannnnnnn nntgg
155010DNAartificial sequenceThis is the restriction cleavage site
for Xmn I 50gaannnnttc 10
* * * * *
References