Note: Descriptions are shown in the official language in which they were submitted.
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DIABETES THERAP~
The present invention relates to gene therapy and
provides stable, transformed m~mm~l ian cell lines and vectors
useful as vehicles for transferring functional DMA se~uences
whose protein products are useful in the treatment of
dia~etes mellitus.
The human hormone glucagon is a 29-amino acid
hormone produced in pancreatic A-cells. The hormone belongs
to a multi-gene family of structurally related peptides that
include secretin, gastric inhibitory peptide, vasoactive
intestinal peptide and glicentin. These peptides variously
regulate carbohydrate metabolism, gastrointestinal mobility
and secretory processing. However, the principal recognized
actions of pancreatic glucagon are to promote hepatic
glycogenolysis and gluconeogenesis, resulting in an elevation
of blood sugar levels. In this regard, the actions of
glucagon are counter regulatory to those of insulin and may
contribute to the hyperglycemia that accompanies diabete~
mellitus (Lund, P.K., et al., Proc. Natl. Acad. Sci. U.S.A.,
79:345-349 (1982)).
When glucagon binds to its receptor on insulin
producing cells, cAMP production increases which in turn
stimulates insulin expression (Korman, L.Y., et al.,
Dia~etes, 34:717-722 ~1985)). Moreover, high levels of
insulin down-regulate glucagon synthesis by a feedback
inhibition mechanism (Ganong, W.F., Review of Medical
P~ysiology, Lange Publications, Los Altos, California, p. 273
(1979)). Thus, the expression of glucagon is carefully
regulated by insulin, and ultimately by serum glucose levels.
Preproglucagon, the precursor form of glucagon, is
encoded by a 360 base pair gene and is processed to form
-~ proglucagon ~Lund, et al., Proc. Natl. Acad. Sci. U.S.A.
7g:345-349 (1982~). Patzelt, et al. (Nature, 282:260-266
~1979)) demonstrated that proglucagon is further processed
into glucagon and a second peptide. Later experiments
demonstrated that proglucagon is cleaved carboxyl to Lys-Arg
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or Arg-~rg residues (Lund, P.K., et al., Lopez L.C., et al.,
Proc. Natl. Acad. Sci. U.S.A., 80:5485-548g (1983), and sell,
G.I., et al., Nature 302:71~-718 (1983)). Bell, G.I., et
al., also discovered that proglucagon contained three
discrete and highly homologous peptide regions which were
designated glucagon, glucagon-like peptide 1 (GLP-l), and
glucagon-llke peptide 2 (GLP-2). Lopez, et al., demonstrated
that GLP-l was a 37 amino acid peptide and that GLP-2 was a
3~ amino acid peptide. Analogous studies on the structure of
rat preproglucagon revealed a similar pattern of proteolytic
cleavage at Lys-Arg or Arg-Arg residues, resulting in the
formation of glucagon, GLP-l, and GLP-2 (Heinrich, G., et
a7., Endocrinol., 115:2176-2181 ~1984)). Finally, human,
rat, bovine, and hamster se~uences of GLP-l have been found
to be identical ~Ghiglione, M., et al., Diabetologia, 27:599-
6~0 (1984)).
The conclusion reached by Lopez, et al., regarding
the size of GLP-l was confirmed by studying the molecular
forms of GLP-l found in the human pancreas (Uttenthal, L.O.,
et a7. J. Cli~. Endocrinol. Metabol., 61:472-479 (1985)).
Their research showed that GLP-l and GLP-2 are present in the
pancreas as 37 and 34 amino acid peptides respectively.
The similarity between GLP-l and glucagon suggested
to early investigators that GLP-l might have biological
activity. Although some investigators found that GLP-l could
induce ra~ brain cells to synthesize cAMP (Hoosein, N.M., et
al., Febs Lett. 178:83-86 ~1984)), other investigators failed
to identify any physiological role for GLP-l (Lopez, L.C , et
a7. supra). The failure to identify any physiological role
for GLP-l caused some investigators to question whether GLP-l
was in fact a hormone and whether the relatedness between
glucagon and GLP-l might be artifactual.
It has now been shown that biologically processed
forms of ~LP-l have insulinotropic properties and delay
gastrlc emptying. GLP-1(7-34) and GLP-1(7-35) are disclosed
in U.S. Patent No: 5,118,666, herein incorporated by
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reference. GLP-1(7-37) is disclosed in U.S. Patent No:
5,120,712, herein incorporated by reference.
Variants and analogs of GLP-1 are known in the art.
These variants and analogs include, for example, GLP-1(7-36),
Gln9-GLP-1(7-37), Thr16-Lys18-GLP-1(7-37), and Lys18-GLP-1(7-
37). Derivatives of GLP-1 include, for example, acid
addition salts, carboxylate salts, lower alkyl esters, and
amides (see, e.g., WO91/11457). Generally, the various
disclosed forms of GLP-1 are known to stimulate insulin
lC secretion (insulinotropic action) and cAMP formation (see,
e.g., Mojsov, S., Int. ~. Peptide Protein Research, 40:333-
343 (1992)).
More importantly, numerous investigators have
demonstrated a predictable relationship between various in
vi~ro laboratory experiments and mammalian, especially human,
insulinotropic responses to exogenous administration of GLP-
1, GLP-1(7-36) amide, and GLP-1(7-37) acid (see, e.g., Nauck,
M.A., et a7., Diabetologia, 36:741-744 (1993); Gutniak, M.,
et al., New England J. o~ Medicine, ~2~(20):1316-1322 (1992);
Nauck, M.A., et al., ~. Clin. Invest., ~1:301-307 (1993); and
Thorens, B., et al., Diabetes, 42:1219-1225 (1993)).
The fundamental defects responsible for causing
hyperglycemia in mature onset diabetes include impaired
secretlon of endogenous insulin and resistance to the e~fects
o~ insulin by muscle and liver tissue ~Galloway, J.S.,
Dia~etes Care, 13:1209-1239, (1990)). The latter de~ect
results in excess glucose production in the liver. Thus,
whereas a normal individual releases glucose at the rate of
approximately 2 mg/kg/minute, a patient with mature onset
diabetes releases glucose at a rate exceeding 2.5
mg~kg/minute, resulting in a net excess o~ at least 70 grams
o~ glucose per 24 hours.
Because there exists exceedingly high correlation
between hepatic glucose production, fasting blood glucose
levels, and overall metabolic control as indicated by
glycohemoglobin measurements (Galloway, J.A., supra; and
Galloway, J.A., et al., Clin. Therap., 12:460-472 (1990)), it
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is readily apparent that control of fasting blood glucose is
essential for achieving overall normalization of metabolism
sufficient to prevent hyperglycemic complications. Since
existing insulin therapies rarely normalize hepatic glucose
production without producing significant hyperinsulinemia and
hypoglycemia (Galloway, J.A., and Galloway, J.A., et al.,
supra) alternative approaches for diabetic therapy are
needed.
Therapy based on administration of longer acting
10- GLP-l analogs is one such approach. To date however, this
approach has failed to deliver long term e~ficacious doses to
individuals due in large part because the serum hal~ e o~
GLP-1(7-37) is ~uite short. There~ore, the quest for
alternative approaches continues.
Gene therapy o~fers a new avenue ~or treating
diseases rooted in hormone de~iciencies because it operates
as an i~ vivo protein production and delivery system. This
is an especially attractive approach since gene therapy also
offers the possibility of physiologically regulating the
2~ production and secretion o~ proteins in response to
homeostatic mediators within the body.
Gene therapy can be effected in a number of ways.
~etroviral-mediated gene transfer was suggested ~or treating
human diseases involving mal~unctioning bone marrow.
Anderson et al., Science 226: 401 (1984). In addition, PCT
Publication Number W093/09222 (May 13, 1993) and U.S. Patent
Number 5,3g9,346 (March 21, 1995) disclose the genetic
alteration of primary human cells that are cultured then
reintroduced into the body for the treatment o~ a variety of
diseases.
Many heritable diseases such as diabetes result
from the absence of a functional gene necessary to provide
the ~n;~l with an ade~uate supply o~ a vital protein. The
goal of gene therapy is to deliver a nucleic acid sequence,
3~ present on an RNA or DNA vector, which is capable of encoding
the desired, therapeutic protein. Although a number of
methods exist to deliver the DMA or RNA vector containing the
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--5--
desired nucleic acid sequence into the target m~mm~l ian
cells, two procedures termed ex vivo and in vivo are
generally employed.
Ex vivo gene therapy consists of four primary
steps: (1) Primary cells (target cells) are removed from the
individual in need of therapy; (2) The gene therapy
nucleotide sequence is incorporated into the target cell in
vl tro; (3~ The transformed cells expressing the incorporated
nucleotide sequence encoding the protein of interest are
identified, isolated, and expanded; and (4) The transformed
cells are reintroduced into the individual.
~ x vivo therapy generally results in the
incorporation of the nucleotide sequence encoding the protein
of interest into the chromosomal DNA of the target cell. The
critical step of ex vivo gene therapy is the proper
introduction of the nucleotide se~uence encoding the desired
protein lnto the target cell This transfer of DNA can be
accomplished by a number of well documented methods such as:
calcium phosphate precipitation, electroporation, and
adenoviral or retroviral vectors (Current Protocols in
Molecular 3iolo~v, John Wiley and Sons, 1989; Methods of Cell
Bioloov, 43: 161 - 189, 1994; Proc. Natl. Acad. Sci. USA 85:
6460 - 6464, 1985); although, other well known methods are
also consistent with this invention.
In vivo gene therapy generally describes the
transfer of a desired nucleic acid sequence, located on a
transport vector, directly into an individual in need of
therapy. This gene therapy approach does not require that
the target cells be ~irst removed and manipulated in vitro.
O~ce the vector containing the desired nucleotide sequence is
introduced into the individual, the vector moves into the
nucleus of the target cell and the nucleotide sequence of
interest integrates into the chromosomal DNA of the target
cell ~ith varying degrees of efficiency. A number of well
documented methods exist for introducing nucleic acids into
an individual requiring therapy such as direct injection of
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D~A and the use o~ recombinant viral vectors (Gene TheraDv, A
Hand~ook for Phvsici~n~, Mary Ann Liebert, Inc., 1994).
The present invention provides a method o~ treating
both Type I and Type II diabetics through a di~erent gene
therapy approach. A stable m~mm~l ian cell line is
trans~ormed by a nucleic acid vector such that it secretes a
GLP-1(7-37)-based protein, as de~ined by SEQ ID NO 1,
followed by implantation into an individual needing
treatment. Once implanted, the G~P-1(7-37)-based protein, in
13 con~unction with high serum glucose levels, causes pancreatic
beta cells to produce insulin in non-insulin dependent
d~abe~es mellitus (NIDDM~ patients and delays gastric
emptying in both NIDDM and insulin dependent diabetes
mel 7itus IDDM patients.
Accordingly, one embodiment o~ this invention
provides a method o~ treating Type I or Type II diabetes in a
mammal in need thereo~ comprising implanting a cell line
trans~ormed with a vector comprising a promoter driving
expression of a DNA se~uence encoding a protein o~
SEQ ID NO 1
His-Xaa1-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-
Leu-Xaa2-Gly-Gln-Ala-Ala-Xaa3-Xaa4-Phe-Ile-Ala-Trp-Leu-
Val-Lys-Gly-Arg-Xaa5 (gs~ ID NO 1)
wherein
Xaa1 is Ala, Gly, Val, Thr, and Ile;
Xaa2 is Glu, Gln, Ala, Thr, Ser, and Gly;
Xaa3 is Lys, and Arg;
Xaa4 is Glu, Gln, Ala, Thr, Ser, and Gly; and,
Xaa5 is Gly-OH or is absent;
3~ into said m~mm~ I such that it is ;mmllnologically isolated
~rom the mammal's immune system and secretes a protein o~ SEQ
ID NO 1 into said patient.
Pre~erred proteins o~ SEQ ID Mo 1 are those in
which Xaal is Ala, Xaa2 is Glu, Xaa3 is ~ys. Xaa4 is Glu, and
Xaa5 is Gly-OH. More pre~erred are those in which Xaa1 is
Ala, Gly, Val, Thr, and Ile; Xaa2 is Glu, Gln, Ala, Thr, Ser,
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and Gly; Xaa3 is Lys, and Arg; Xaa4 i5 Glu, Gln, Ala, Thr,
Ser, and Gly; and, Xaa5 is Gly-OH or is absent. Another
pre~erred group are those in which Xaa1 is Val and Xaa3 is
Lys. A ~urther pre~erred group o~ proteins are those wherein
Xaa1 is Ala or Val, Xaa2 Glu, Xaa3 is Lys or Arg, Xaa4 is
Glu, and Xaa5 Gly-OH or is absent. Yet another pre~erred
group are those in which Xaa1 is Ala, Xaa2 Glu, Xaa3 is Lys,
Xaa4 is Glu, and Xaa5 Gly-OH. Still another pre~erred group
is when Xaa1 is Val, Xaa2 Glu, Xaa3 is Lys, Xaa4 is Glu, and
Xaa5 Gly-OH.
Nucleotide sequences encoding any one o~ the
polypeptides o~ SEQ ID NO 1 may be prepared by a variety of
means readily apparent to those skilled in the art. Wholly
synthetic nucleotide sequences or semi-synthetic sequences
deri~ed in part ~rom a natural GLP-1 gene may be used. Owing
to the natural degeneracy o~ the genetic code, the skilled
artisan will recognize that a sizable yet de~inite number of
nucleotide sequences may be constructed which encode the
proteins o~ SEQ ID MO 1. A synthetic DNA sequence encoding a
G~P-1-based protein o~ SEQ ID NO 1 may be prepared by
techni~ues well known in the art in substantial accordance
wit~ the teachings o~ Brown, ~ ~1. (1979) Methods in
~zymo70~y, Academic Press, N.Y., Vol. 6~, pgs. 109-151. The
DNA seq~ence may be generated using conventional DNA
synthesizing apparatus such as an Applied Biosystems Model
380A or 380B DNA synthesizer (commercially available ~rom
Applied Biosystems, Foster City, Cali~ornia). Commercial
services are also available for the construction of such
nucleotide sequences based on the amino acid sequence.
In one pre~erred embodiment o~ the invention as
exempli~ied herein, the coding sequence ~or a protein o~ SEQ
~D N~ 1 is the ~ollowing:
5' - CAT GCT GAA GGG ACC TTT ACC AGT GAT GTA AGT TCT TAT TTG
GAA GGC CAA GCT GCC AAG GAA TTC ATT GCT TGG CTG GTG AAA
35 GGC CGA GGA - 3' ~SEQ ID NO 2).
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This sequence encodes the following protein o~ SEQ ID NO 1:
H2N- His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly -OH (SEQ ID NO 3).
In another preferred embodiment of the invention as
- exempli~ied herein, the coding se~uence ~or a protein of SEQ
ID NO 1 is the following:
5 ' - CAT GTT GAA GGG ACC TTT ACC AGT GAT GTA AGT TCT TAT ~TG
GAA GGC CAA GCT GCC AAG GAA TTC ATT GCT TGG CTG GTG AAA
GGC CGA GGA - 3~ (SEQ ID No 4).
This sequence encodes the following protein of SEQ ID NO 1:
H2N- His Val Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu
Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys
Gly Arg Gly -OH (SEQ ID NO 5).
15_ Coding regions for SEQ ID NO 1 may be fused to a
leader sequence which signals the cell to secrete a precursor
peptide of SEQ ID NO 1 that is subsequently processea by the
cell during the secretion process to a protein of SEQ ID NO
l Many such leader sequences are know in the art. One well
known and preferred leader sequence is the hybrid tissue
plasminogen activator/protein C prepropeptide described in
serg et a 7 . Biochem. Biophys. Res. Commun. 179: 1289-1296
~1991). Typically nucleotide se~uences encoding precursor
peptides of SEQ ID NO 1 are flanked by linker DNA to
facilitate enzymatic ligation into expression vectors as is
later exemplified herein.
Once a suitable coding sequence of SEQ ID N~ 1 iS
constructed and optionally fused and flanked by an
apprcpriate leader sequence and linker DNA, the construct is
ligated into an expression vector which is then introduced
into an ap~L ~L iate cell line. Construction of suitable
vectors containing the desired coding and control sequences
may be constructed by standard ligation techniques. Isolated
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plasmids or nucleotide ~ragments are cleaved, tailored, and
religated in the manner necessary to achieve the desired
plasmids.
To ef~ect the expression of a polypeptides o~ SEQ
ID NO 1, one ligates a nucleotide se~uence encoding the
polypeptide into an appropriate recombinant nucleotide
expression vector through the use o~ appropriate enzymes.
The nucleotide sequence encoding a polypeptide o~ SEQ ID NO 1
is designed to possess restriction endonuclease cleavage
sites a~ either end of the DNA to ~acilitate isolation from
and integration into these ampli~ication and expression
plasmids. The coding sequence may be readily modi~ied by the
use of synthetic linkers to facilitate the incorporation of
the coding sequence into the desired cloning vectors by
techniques well known in the art. The particular
en~onucleases employed will be dictated by the restriction
endonuclease cleavage pattern of the parent expression vector
to be employed. The choice of restriction sites are chosen
so as to properly orient the coding sequence such that it is
properly associated with the promoter and ribosome binding
site of the expression vector, both o~ which are ~unctional
in the host cell in which a compound of SEQ ID NO 1 is to be
ex~ressed.
In addition to the desired nuceotide sequence which
will code for the therapeutic protein, the expression vector
m~y contain several other ~unctional elements. One such
element is the promoter and upstream regulatory sequences
which control the level of expression of the protein o~
interest. Some expression vectors contain promoters and
regulatory sequences which normally regulate transcription o~
cellular genes. One such promoter is the mouse
metallothionein-I promoter which has been shown to function
both in-vitro and in-vivo (Palmiter et al., Nature 300: 611 -
615, 1982). In addition, promoters and regulatory sequences
from viruses are ~requently used in expression vectors
~Dijkema et al., ~MBO J. 4, 471, 1985; Gorman et al., Proc.
Natl. Acad. ~ci. 79: 6777, 1982; Boshart, et al. Cell 41:
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-10 -
521, lg85). Although, Verma et al. have shown that the
retrovirus promoter and enhancer se~uences do not ~unction
for long periods of time in-vivo. In addition, the
expression vector also carries an origin of replication as
well as marker se~uences which are capable o~ providing
phenotypic selection in transformed cells.
Because the proteins useful in the present
invention do not re~uire post-translational processing
mechanisms other than enzymatic removal of the propeptide
leader sequence, many stable human cell lines are consistent
with the practice of the invention. One preferred cell line
i~ the human embryonal kidney cell line 293, available from
the permanent collection of the American Type Culture
~ollection.
A number of well known methods exist for
introducing the genetic material into target cells such as
chemical ~calcium phosphate precipitation), physical
(electorporation and microin~ection), and viral methods
~adenovirus, retrovirus, and adeno-associated virus) (Methods
~or Gene Trans~er, Gene Thera~v, Mary Ann Liebert, Inc.,
lg~4). All such methods are consistent with the practice of
the prese~t invention. The techniques o~ transforming
mammalian cells with the aforementioned vector types are well
known in the art and may be found in such general references
as Maniatis, et al. (1989) Molecul~r Cloninc: A Laboratory
M~nll~l, Cold Spring Harbor Press, Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York or Current Protocols
in Molecular Bioloov, Vol. 1, (1988), Wiley Interscience,
and supplements.
3~ Stable transformed cell lines that express proteins
Q:E SE~ ID NO 1 must then be implanted into the individual in
need of such treatment. Because such transformed cell lines
generally will be histologically incompatible with the
indivi~uals receiving them, the cells must to be protected
from the recipient's immune system. Once way o~ protecting
the implanted cells is by masking them with F(abl)2 fragments
speci~ic ~or HLA class I antigens. Immunological masking
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-11-
methods are well known in the art. For example, see Faust et
al. ~cience 252:1700-1702 (1991). Other means for protecting
the implanted cells from the recipient's immune system are
consistent with this invention. Such methods include but are
not limited to encapsulation in semi-permeable membranes
(Lanza et al. Diabetes 41: 1503 - 1510) and through the use
o~ immunosuppressants (Rynasiewicz et al., Diabetes 31: 92 -
108, 1982).
By way o~ illustration, the ~ollowing examples are
provided to help describe how to make and practice the
various embodiments of the invention. These examples are in
no way meant to limit the scope o~ the invention.
~i~Amr~le
Construction of Intermediate
Plasmid ~LP53-tLB+~TP-1
A. Pre~aration of B~lTI-Muna sean-AvrII Diaested ~P53-tLB
The plasmid pLP53-tLB was isolated ~rom E. coli K12
AG1 lon deposit under terms of the Budapest Treaty and made
part of the permanent stock culture collection of the
Northern Regional Research Laboratories (NRRL), agricultural
Research Service, U.S. Dept. of Agriculture, Peoria, IL 61604
under accession number NRRL B-18714) using the Plasmid
Puri~ication Midi Kit (Qiagen, Inc., 9600 DeSoto Avenue,
Chatsworth, CA 91311).
Sixty ~l (approximately 20 ~g) o~ pLP53-tLB DNA was
digested with 2 ~l (20 units) of ~glII in a 70 ~1 reaction
volume containing 50mM Tris-HCl (pH 8.0), 10mM MgCl2, and
l~M NaCl. The sample was incubated at 37~C for one hour.
17.5 ~l of 5x stop mix (25% glycerol, 2% SDS, 0.05%
bromophenol blue, 0.05% xylene cyanol in water) was added and
then the reaction was heated at 70~C for 15-20 minutes to
inactivate the restriction enzyme. The mixture was spin
dialyzed using G-50 Sephadex Quick Spin columns (Boehringer
Mannheim Corporation, P.O. Box 50414, 9115 Hague Road,
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WO97/29180 PCT~US97/01978
-12-
Indianapolis, IN 46250-0414) to remove the reaction
components.
The 5' protruding ends created by cleavage with
~glII were removed using Mung Bean Nuclease. The BalII
digested pLP53-tLB DNA was incubated with 0.3 ~l
(approximately 3.3 units) of Mung Bean Nuclease in a 100 ~l
reaction volume containing 10mM Tris-HCl (pH 7.9 at 25~C),
lOmM MgC12, 50mM NaCl, lmM DTT, and lmM ZnS04. The reaction
was allowed to proceed for 3D minutes at 30~C. One ~l of 1%
~DS was added to inactivate the nuclease. Due to an
- incomplete BalII digest, the digested and undigested DNA was
separated by gel electrophoresis. Ten ~l o~ gel loading dye
(0.25% bromophenol blue, 0.25~ xylene cyanol, 30% glycerol in
water) was added to the reaction. The reaction was loaded
into the preparative well of a 1.5% NuSieve GTG agarose (EMC
Bioproducts, 191 Thomaston Street, Rockland, ME 04841)/TAE
buf~er gel and then electrophoresed. The gel was stained
with ethidium bromide and the DNA wa$ visualized by
ultraviolet light. The digested DMA band was excised with a
scalpel and placed into two micro-tubes. The DNA was
purified from the low melting point agarose usiny the Wizard
PCR Preps DNA Purification System (Promega, 2800 woods Hollow
Road, Madison, WI 53711-5399).
One hundred ~1 of BalII-Mung Bean digested pLP53-
tLB DNA was further digested with 4 ~l (approximately 16
~ units) of AvrII in a reaction volume of 120 ~1 containing
lOmM Tris-HCl (pH 7.9 at 25~C~, 10mM MgC12, 50mM NaCl, lmM
DTT . The sample was incubated at 37~C for 30 minutes. To
prevent recircularization, the B~lII-Mung Bean-AvrII digested
pLP53-tLB DNA was dephosphorylated (removal of 5~ phosphate
groups) by the addition of 2 ~l (2 units) of calf intestinal
alkaline phosphatase to the reaction. The sample was
incubated at 37~C for an additional 30 minutes. Twenty-four
~l of 5x stop mix was added. The sample was heated at 70~C
for 15-20 minutes to inactivate the enzymes and then spin
dialyzed using G-50 Sephadex Quick Spin columns (Boehringer
Mannheim Corporation, P.O. Box 50414, 9115 ~ague Road,
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W O 97/29180 PCT~US97/01978
Indianapolis/ IN 46250-0414) in order to remove the ~II
produced small DNA fragments. The DNA was precipitated by
addition o~ O.1 volume oE 3M sodium acetate (pH 5.2) and 2.5
volumes of absolute ethanol. This mixture was mixed
5 thoroughly and then chilled to -20~C. The precipitate was
coll ected by centrifugation for 30 minutes. The supernatant
was discarded and the pellet was washed with 700 ~l of cold
70% ethanol. The sample was centrifuged for 15 minutes. The
supernatant was discarded, the DNA pellet was dried and then
10 resuspended in 25 ,Ul of water.
B. Pre~ration of GLP-l Linker
The following single stranded DNA segments were
conventionally synthesized by methods well known in the art
15 on an automated DNA synthesizer (model 394 Applied Biosystems
850 Lincoln Center Drive, Foster City, CA 94404-1128) using
~-cyanoethyl phosphoramidite chemistry.
(~T,P--1 . 1
20 5' - GACATGCTGA AGGGACCTTT ACCAGTGATG TAAGTTCTTA TTTGGAAGGC
CAAGCTGCCA AGGAATTCAT TGCTTGGCTG GTGAAAGGCC GAGGATAGGG
ATCCC - 3' (SEQ ID NO 6)
GLP-1.2
25 5' - CTAGGGGATC CCTATCCTCG GCCTTTCACC AGCCAAGCAA TGAATTCCTT
GGCAGCTTGG CCTTCCAAAT AAGAACTTAC ATCACTGGTA AAGGTCCCTT
CAGCATGTC - 3' (SEQ ID NO 7)
GLP-l.l and GLP-1.2 are complementary DNA
30 molecules. The synthetic DNA moIecules were dissolved in
water and stored at less than 0~C.
To anneal the DNA strands, approximately 92.7
pmoles each of GLP-l.l and GLP-1.2 were mixed in 50mM Tris-
HCl (pH 7.4) and lOmM MgC12 in a total volume of 80 111 and
35 boiled for~5 minutes. The mixture was slowly brought to room
temperature and then transferred to 4~C overnight. This
process allowed the two complementary strands to anneal and
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- -14-
form the double stranded DNA linker known as GLP-1. The
linker was stored at -20~C. In order to be able to ligate
into the dephosphorylated ~g1II-Mung Bean-AvrII digested
pLP53-tLB DNA segment, the GLP-1 linker must have 5'
phosphate groups. The phosphate groups were added by the use
of the enzyme T4 polynucleotide kinase. The kinase reaction
contained 80 ~l of the GLP-1 linker, 0.33~M ATP, 70mM Tris-
HCl (pH 7.6), 10mM MgCl2, 100mM KCl, lmM ~-mercaptoethanol
and 37.2 ,ul t372 units) of T4 polynucleotide kinase. The
reaction was incubated at 37~C ~or 30 minutes. Sixteen ~l of
500mM EDTA was added to stop the reaction. The reaction was
extracted once with a mixture of phenol:chloroform:isoamyl
alcohol (25:24:1) followed by an extraction with
chloroform:isoamyl alcohol (24:1). One hundred ~l of the
a~ueous layer was spin dialyzed using G-50 Sephadex Quick
Spin columns (Boehringer Mannheim) in order to remove the
reaction components. The DNA was precipitated by addition of
0.1 volume o~ 3M sodium acetate (pH 5.2), 0.1 volume of 100mM
MgCl2 and 2.5 volumes of absolute ethanol. This mixture was
2G mixed thoroughly and then chilled at -20~C. The precipitate
was collected by centrifugation for 30 minutes. The
supernatant was discarded and the pellet was washed with 700
~l of cold 70% ethanol. The sample was centrifuged for 15
minutes. The supernatant was discarded, the DNA pellet was
dried and then resuspended in 10 ~l of water.
C. Final Construction of ~TP53-tLB+GLP-1
The DNA prepared in Example lA was ligated with
linker GLP-1. Two ~l of DNA prepared in Example lA and 4 ~l
o~ GLP-1 linker were ligated in a reaction that contained 2
,Ul (2 units) of T4 DNA ligase, 50mM Tris-HCl (pH 7.6), 10mM
MgC12, lmM ATP, lrnM DTT, and 50% (w/v) polyethylene glycol-
8000 in a total volume of 10 ~l. The mixture was incubated
at 16~C for 16 hours. The ligation was used to transform E.
coli K12 I~VaF' cells as generally described below.
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D. Tr~nsformation Procedure
Frozen competent E. coli K12 INVaF' cells were
= obtained from Invitrogen (3985 B Sorrento Valley Boulevard,
San Diego, CA 92121). Two ~l of 0.5M ~-mercaptoethanol were
added to 50 ~l of thawed competent cells. About 1-2 ~l of
the ligation reaction was mixed with the cells. The cell-DNA
mixture was incubated on ice for 30 minutes, heat-shocked at
42~C for exactly 30 seconds and then chilled on ice for 2
minutes. The cell-DNA mixture was diluted into 450~1 of SOC
media (2% tryptone, 0.05% yeast extract, lOmM NaCl, 2.5mM
KCl, lOmM MgCl2, lOmM MgSO4, 20mM glucose in distilled water)
and lncubated at 37~C for one hour in a rotary shaker set at
about 225 rpm. Aliquots o~ up to 200 ~l were plated on TY-
agar plates ~1% tryptone, 0.5% yeast extract, 1% NaCl, and
1.5% agar, pH 7.4) containing lOO~g/ml ampicillin and then
incubated at 37~C until colonies appear.
E. DNA Tsolation
Following transformation, ampicillin resistant
cells were picked and inoculated into 3 ml of TY broth (1%
tryptone, 0.5~ yeast extract, 1% NaCl, pH 7.4) cont~;n;ng
lOO~g/ml ampicillin. These cultures were grown for about 16
hours at 37~C with aeration. Plasmid DNA was isolated from
cultures using Wizard Minipreps obtained from Promega (2800
Woods Hollow Road, Madison, WI 53711-5399). Recombinant
plasmids were identified by digestion with restriction
endonucleases followed by gel electrophoresis analysis.
To obtain larger amounts of pLP53-tLB+GLP-1 plasmid
DNA, large scale isolation was performed using the Plasmid
Purification Midi Kit (Qiagen, Inc.).
Exam~le 2
Construction of DGT-h+tLB+GLP-1
A. Preparation of BclI Diaested DGT-h
The plasmid pGT-h was isolated from ~. coli K12 GM48
(on deposit under terms of the Budepest Treaty and made part
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- of the permanent stock culture collection of the NRRL under
accession number B-18592 using the Plasmid Puri~ication Midi
Kit (Qiagen, Inc.).
Ten ~g (37.5 ~l) of pGT-h DNA was digested to
completion with 2 ~1 (20 units) of ~clI in a 45 ~l reaction
volume containing 50mM Tris-HCl (pH 8.0), lOmM MgCl2, 50mM
NaCl. The sample was incubated at 50~C for 1 hour. Eleven
~l of 5x stop mix was added to the reaction mixture. The
mixture was heated at 70~C for 15-20 minutes to inactivate
the restriction enzyme and then spin dialyzed using G-50
Sephadex Quick Spin columns (Boehringer Mannheim) in order to
remove the reaction components. The DNA was precipitated by
adding 0.1 volume of 3M sodium acetate (pH 5.2) and 2.5
volumes of absolute ethanol. This mixture was mixed
thoroughly and then chilled at -20~C. The precipitate was
collected by centrifugation for 30 minutes. The supernatant
was discarded and the pellet was washed with 700 ~l of cold
70% ethanol. The sample was centrifuged for 15 minutes. The
supernatant was discarded, the DNA pellet was dried and then
resuspended in 20 ~1 o~ water.
Calf intestinal alkaline phosphatase was used to
remove the 5' phosphate groups from the DNA segment in order
to prevent recircularization of the pGT-h. Ten ~l of the
BclI digested pGT-h DNA was treated with 1 ~1 ~1 unit) of
calf intestinal alkaline phosphatase in a 15 ~l reaction
cont~;ning 50mM Tris-HCl (pH 8.5 at 20~C) and O.lmM EDTA.
The reaction was allowed to proceed for 45 minutes at 37~C.
The phosphatase was inactivated by the addition of 1~1 of
500mM EDTA and then heating at 65~C for 10 minutes. The
reaction volume was increased to 100~1 with water and then
extracted twice with phenol:chloroform:isoamyl alcohol
(25:24:13 followed by extraction with chloroform: isoamyl
alcohol (24:1). The DNA was recovered from the a~[ueous layer
by the addition of 0.1 volumes o~ 3M sodium acetate (pH 5.2)
and 2.5 volumes of absolute ethanol. The mixture was mixed
thoroughly and then chilled at -20~C. The precipitate was
collected by centrifugation for 30 minutes. The supernatant
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-17-
was discarded and the pellet was washed with 700 ~l of cold
70% ethanol. The sample was centrifuged for 15 minutes. The
supernatant was discarded, the DNA pellet was dried and then
resuspended in 25 ~l of water.
B. Pre~aration of ~LP53-tLB+GLP-l BamHI Fra~ment
Thirty-five ~1 (10.6 ~g) of pLP53-tLB+GLP-l DNA,
prepared in Example 1, was digested with 0.5 ~l (25 units) of
BamHI in a 40 ~1 reaction volume containing 50mM Tris-~Cl (pH
8.0), 10mM MgCl2, and 100mM NaCl. The reaction was allowed
to proceed at 37~C for one hour. Five ~l of gel loading dye
was added to the reaction. The reaction was loaded into the
preparative well of a 4% NuSieve GTG agarose/TAE bu~er gel.
The DNA was electrophoresed for about one hour at 70 constant
volts. The gel was stained with ethidium bromide and then
the DNA was visualized by ultraviolet light. The desired 213
base pair DNA band was excised using a scalpel. The DNA was
purified from the low melting point agarose using Wizard PCR
preps (Promega).
C. Final Construction o~ ~GT-h+tLB+GLP-l
The DNA prepared in Example 2A was ligated with DNA
prepared in Example 2B. One ~l of DNA from Example 2A and
10.5 ~1 of DNA ~rom Example 2B were ligated in a reaction
that contained 2 ~1 (2 units) of T4 DNA ligase, 50mM Tris-HCl
25 (pH 7.6), 10mM MgCl2, lmM ATP, lmM DTT, and 50% (w/v)
polyethylene ~lycol-8000 in a total volume of 20 ~l. The
mixture was incubated at 16~C for 16 hours. The ligation
reaction was used to trans~orm E. coli K12 INVo~F~ as
described in Example lD. Plasmid DNA was isolated from
ampicillin resistant cultures as described in Example lE.
To obtain larger amounts o~ pGT-h+tLB+GLP-l plasmid
DNA ~or the purpose o~ transfection of m~mm~l ian cells, large
scale isolation was performed using the alkaline lysis
method.
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Ex~m~le 3
Construction o~ ~GT-h+tTB+V~l8G~P-l
The plasmid pGT-h+tLB+Val8GLP-1 was constructed
substantially in accordance with Examples 1 and 2. To
accommodate the change in the amino acid se~uence (Ala to val
at Xaa1 in SEQ ID NO 1) the following coding se~uences were
substituted for those described in Example ~B-
V~18GLP-1.1
5~ - GACATGTTGA AGGGACCTTT ACCAGTGATG TAAGTTCTTA TTTGGAAGGC
CAAGCTGCCA AGGAATTCAT TGCTTGGCTG GTGAAAGGCC GAGGATAGGG
ATCCC - 3' (~EQ ID NO 8)
V~18~T,P-1.2
5' - CTAGGGGATC CCTATCCTCG GCCTTTCACC AGCCAAGCAA TGAATTCCTT
GGCAGCTTGG CCTTCCAAAT AAGAACTTAC ATCACTGGTA AAGGTCCCTT
CAACATGTC - 3~ (SEQ ID NO 9)
~ le 4
Construction of Tntermediate
Plasmid pM100-neo
A. Pre~aration of ~coRI Diaested ~M100
The plasmid pM100 (pOK12) was isolated from E. coli
K12 RRl~M15 usiny Magic Minipreps (Promega). See Vieira and
Messing, Gene 100:189-94, 1991). Forty ~l of pM100 DMA was
digested to completion with 3 ,Ul of EcQRI in a reaction
volume of 50 ~l containing 50mM Tris-HCl (pH 8.0), lOmM MgCl2,
and lOOmM NaCl. The sample was incubated at 37~C for 2
hours. The digested DNA was precipitated with ethanol. The
final DNA pellet was resuspended in 30 ~1 of water.
B. Pre~aration of ~BK-neo ~coR:r Fra~rment
The plasmid pBK-neo 1 (described in U S Patent No:
5,550,036, herein incorporated by reference, and available
from the American Type Culture Collection under terms of the
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--19--
Budapest Treaty via Accession Mo Atcc 37224) was isolated
from E. coli K12 HB-1 using the large scale alkaline lysis
~ method. Three ~l (26.3 ~g) of pBK-neo DNA was digested to
completion with 7 ~l (15 units) of ~_RI in a reaction volume
of 200 ~l containing 50mM Tris-HCl (pH 8.0), lOmM MgCl2, and
lOOmM NaCl. The sample was incubated at 37~C. Fifty ~l o~
the reaction was loaded into the preparative well o~ an
agarose/TAE bu~er gel and then electrophoresed. The desired
4.2 Kb DNA band was isolated ~rom the agarose by
electrophoresing onto DEAE-cellulose membrane. Following
elution from the DEAE-cellulose membrane, the DMA was
precipitated with ethanol. The final DNA pellet was
resuspended in 30 ~l of water.
C. Final Construction o~ ~M100-neo
The DNA prepared in Example 4A was ligated with DNA
prepared in Example 4B. Two ~l of DNA from Example 4A and 2
~l of DNA from Example 4B were ligated in a reaction that
contained 1 ~l (1 unit) of T4 DNA ligase, 50mM Tris-HCl ~pH
7.6), lOmM MgCl2, lmM ATP, lmM DTT, and 50% (w/v)
polyethylene glycol-8000 in a total volume of 16 ~l. The
mixture was incubated at 16~C for 16 hours. Ten ~l o~ the
ligation reaction was used to transform E. coll K12 RRl~M15
as described in Example 5D. Aliquots of up to 200 ~l were
plated on TY-agar plates (1% tryptone, 0.5% yeast extract, 1%
NaCl, and 1 5% agar, pH 7.4) containing kanamycin and then
incubated at 37~C until colonies appear.
D. DNA Isol~t~on
Following transformation, kanamycin resistant cells
were picked and inoculated into 5 ml of TY broth (1%
tryptone, 0.5% yeast extract, 1% NaCl, pH 7.4) containing
kanamycin. These cultures were grown for about 8 hours at
37~C with aeration. Plasmid DNA was isolated from cultures
using Magic Minipreps obtained from (Promega). Recombinant
plasmids were identified by digestion with restriction
endonucleases ~ollowed by gel electrophoresis analysis
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-20-
To obtain larger amounts of pM100-neo plasmid DNA,
large scale isolation was performed using the Plasmid
Purification Kit (Qiagen, Inc.).
~xam~le 5
~onstruction of ~MT-h+tLB+Val8GLP-l
A. Pre~aration of ~stllO7I-BclI Diaested ~GT-h
The plasmid pGT-h was isolated as described in
substantial accordance with Example 2A. Three ~g (~.6 ~1) of
pGT-h DNA was digested to completion with 1 ~1 (8 units) of
~1107I in a 10 ~1 reaction volume containing lOmM Tris-HCl,
lOmM MgCl2 , lOOmM KCl (pH 8.5 at 37~C). The sample was
incubated at 37~C ~or 1 hour. The BstllO7I digested pGT-h
DNA was purified from the reaction components using the
Wizard DNA Clean-Up System (Promega). The stllO7I digested
pGT-h DNA was concentrated in a 6.7 ~l volume using a
microcon 50 (Amicon, Inc. 72 Cherry Hill Drive, Beverly, MA
01915).
The 6.7 ~1 of ~g~1107I digested pGT-h DNA was
further digested with 1 ~l (10 units) of ~lI in a reaction
volume of 10 ~1 containing 50mM Tris-HCl (pH 8.0), lOmM
MgC12, 50mM NaCl. The sample was incubated at 50~C for 1
hour. To prevent any possible recircularization, the
~stllO7I-BclI digested pGT-h was dephosphorylated (removal of
5' phosphate groups) by the addition of 1 ~l (1 unit) of calf
intestinal phosphatase to the sample. The sample was
incubated at 37~C for 30 minutes. Gel loading dye (0.25%
bromophenol blue, 0.25% xylene cyanol, 30% glycerol in water)
was added to the reaction. The reaction was loaded into the
preparative well of a 1% SeaPlague GTG agarose/TAE bu~fer gel
and then electrophoresed. The gel was stained with ethidium
bromide and then the DNA was visualized by ultraviolet light.
The desired 6.2 Kb DNA band was excised with a scalpel and
placed into a micro-tube. The DNA was puri~ied from the low
melting point agarose using the Wizard PCR Preps DNA
Puri~ication System (Promega).
-
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B. Pre~aration of ~M10~-neo EcoRV-BalII Fra~ment
Five ~g (3.8 ~l) o~ pM100-neo DNA constructed in
Example 4 was digested to completion with 1 ~l (10 units) of
~coRV in a 10 ~1 reaction volume containing 50mM Tris-HCl (pH
8.0), lOmM MgCl2, 50mM NaCl. The reaction was incubated at
37~C for 1 hour. The ~coRV digested pM100-neo was further
digested with 1 ~l (lOunits) of ~glII in a 20 ~l reaction
volume containing 50mM Tris-HCl (pH 8.0), lOmM MgCl2, 50mM
NaCl. The sample was incubated at 37~C ~or 1 hour. Gel
loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 30%
glycerol in water) was added to the reaction. The reaction
was loaded into the preparative well of a 1~ SeaPlaque GTG
agarose/TAE buffer gel and then electrophoresed. The gel was
stained with ethidium bromide and then the DNA was visualized
by ultraviolet light. The desired 1.8 Kb DNA band was
excised with a scalpel and placed into a micro-tube. The DNA
was puri~ied ~rom the low melting point agarose using the
Wizard PCR Preps DNA Purification System (Promega).
C. F;n~l Construction of ~MT-h+tLB+Val8GLP-1
The DNA prepared in Example 5A was ligated with the
DNA prepared in Example 5B and the DNA prepared in Example 3
(pLP53-tLB+Val8GLP~ HI fragment). Four ~l of DNA from
Example 5A, 3 ~l of DNA from Example 5B and 12 ~l of DNA
prepared in Example 3 (pLP53-tLB+Val8GLP-1 E~mHI fragment)
were ligated in a reaction that contained 1 ~l (1 unit) of T4
DNA ligase, 50rnM Tris-HCl (pH 7. 6), lOmM MgCl2, lmM ATP, lmM
DTT, and 50% ~w/v) polyethylene glycol-8000 in a total volume
of 25 ~l. The mixture was incubated at 16~C for 16 hours.
Frozen competent ~. coli K12 DH5a cells were
transformed using about 3-4 ~l of the ligation reaction in
substantial accordance with Example lD, and the plasmid DNA
was isolated in substantial accordance with Example lE.
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-22-
E~mnl e 6
Construction of St~hle Cell lines
Plasmid DNA, either pGT-h+tLB+GLP-l, pGT-
h+tLB+val8GLP-l, or p~T-h+tLB+val8GLP-l was transfected into
human embryonic kidney 293 cells using the stable CaPQ4 method
contained in the Mammalian Transfection Kit available from
Stratagene (11011 Morth Torrey Pines Road, LaJolla, CA
92037). Selection was achieved by the addition o~ 300~g/ml
of hygromycin B (Eli Lilly and Company In~;~n~polis, IN
46285) to the culture medium. Monoclonal cell lines were
expanded and screened ~or the ability to secrete the
corresponding protein of SEQ ID NO 1 into t~e culture medium.
The presence of biologically active GLP-1(7-37)-based protein
in the culture medium was determined by measuring the amount
of luci~erase enzyme present in a biological system that
expressed luci~erase enzyme following stimulation with GLP-l.
~ ~le 7
Im~lantation
The transformed 293 cell were cultured then
surgically transplanted under the kidney capsule o~ 8 week
old zucker Diabetic Fatty (ZDF/GmiTM-~a/~a) male rats. Under
iso~urane anesthesia, a dorsal incision was made just
posterior to the diaphram, and using a rib spreader, the
kidney was exposed. Approximately 20 million trans~ormed 293
cells, in 200 ~l of Hank's buffer, were in~ected just under
the kidney capsule using a 23 gauge blunt needle. The
incision was sutured and protected from chewing with wound
clips .
