Note: Descriptions are shown in the official language in which they were submitted.
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~ TITLE OF THE INVENTION
GENE THERAPY FOR OBESITY
CROSS-REFERENCE TO RELATED APPLICATIONS
Not Applicable
STATEMENT REGARDING FEDERALLY-SPONSORED R&D
Not Applicable
REFERENCE TQ MICROFICHE APPENDIX
Not Applicable
FIELD OF THE INVENTION
This invention relates to methods of gene therapy for
obesity. This invention also relates to vectors u~seful in this gene
therapy.
BACKGROUND OF THE INVENTION
Leptin is a protein expressed by the ob gene. Leptin is
secreted by adipose tissue and appears to be both a satiety factor and a
re~ulator of me~abolism (Levin et al., 1996 P)c~c. Natl Aca~l. Sci. USA
93:1726-1730). Both the mouse gene and its human homologue have
recently been identified and sequenced (Zhang et al., 1994 Natllre
(Londc~n) 372:425-431.)
Mice which are homozygous for the oh gene (c~hlol~) are
obese, perhaps due to an underexpression of leptin. When c~hlc~ mice
are given daily injections of recombinant protein, their food intake was
markedly inhibited and they experienced a reduction in body weight and
fat. In lean (i.e. wild-type) mice, daily injections of leptin lead to
mode.st decreases of food intake and body weight. The results for body
fat have been contradictory. (Pelleymounter et al., 1995, Science
269:540-543; Halaas et al., 1995 Science 269: 543-546; and Campfield et
al., 1995 Sc~ience 269:546-549.
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Obesity in human,s i~s a major di.sorder a.ssociated with mortality, and
may re~sult from a number of causes, and at least some may be due to an
insufficient amount of leptin produced. Since leptin is a protein, and
vulnerable to breakdown and inactivation by the gastrointestinal system,
5 it cannot be delivered orally. It would be desirable to develop a therapy
for leptin delivery for obese patients whose obesity is due, in part to a
paucity of leptin.
Some forms of obesity do not appear to be treatable by the
administration of leptin. In the,se case~s, it is possible that the problem
10 may be due to an insufficient amount of leptin receptors on the cell
~urface. Alternatively, the receptor~ which are pre~ent on the cell
surface may contain mutationls which do not allow them to bind to leptin
efficiently or efficiently process the signal generated by the leptin
binding. Currently no therapy exists which could augment or replace
15 these receptors.
SUMMARY OF THE INVENTION
Not Applicable
20 BRIEF DESCRIPT~ON OF THE INVENTION
This invention i~s related to gene therapy for obesity. One
aspect of thi.s invention involves a method of treating obesity, lowering
serum glucose levels or lowering serum in,sulin levels in a mammal in
need of such therapy comprising delivering a gene encoding an obesity
25 regulating gene to said m~mmal; and allowing sufficient time to pas.s for
transcription and translation of the obesity regulating gene.
Some types of obe~sity are caused by an in,sufficient amount
of leptin or an insufficient amount of functional leptin receptors present
on the cell surface. Thus, another aspect of this invention is a method of
30 treating obesity comprising delivering a gene encoding leptin or a leptin
receptor to a m~mm~l; and allowing sufficient time to pass for
transcription and translation of the leptin or leptin receptor gene.
. . . ~,
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A specific embodiment of this invention is gene therapy for a m~mm~l
whose obesity, elevated level of serum glucose or elevated level of
insulin in due, at least in part to an insufficient amount of leptin
produced. By "insufficient amount of leptin produced" it is envi~ioned
5 that the animal may produce functional leptin, but at lower levels than
required; a complete inability to produce leptin; or production of a
mutated form of leptin which either fuctions less efficienly than native
leptin or does not function at all. Thus, this invention is directed to a
method of treating obesity, an elevated level of serum gluco~se or an
10 elevated insulin~ which is, at least in part, due to an insufficient amount
of leptin produced by a mammal comprising: delivering a gene encoding
leptin to the mammal and allowing suffient time to pass for tran~cription
and translation of the leptin gene.
Obesity may occur in an ;lnim~l which i~s producing norrnal
15 quantities of leptin, but whose leptin receptors are either not able to
bind and process the leptin properly, or have not been produced in
sufficient quantity. These situation.s may be remedied by gene therapy
using a leptin receptor. Thus another aspect of this invention is a
treatment for obesity, exce,ss plasma insulin levels or excess plasma
20 glucose levels~ any of which are a result, at least in part. of an
insufficient amount of functional leptin receptor production by a
mamrnal. Thus, one aspect of this invention i.~ a method of increasing
the amount of leptin receptors in a mammal comprising: delivering a
gene encoding a leptin receptor to the m~mmal, and allowing sufficient
2~ time to pass for transcription and translation of the leptin receptor gene.
BRIEF DESCRIPTION OF THE DRAWTNGS
Figure I is a photograph of two ohlob mice. The mouse on
the right is from a control group. The mouse on the left received gene
30 therapy in accordance with this invention.
Figure 2 is a graph showing the body weight changes of
mice treated with recombinant hurman leptin protein. Injections of
human recombinant leptin were given daily IP, at I ,ug/gm body weight.
Arrows indicate bleeding points.
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Figure 3 is a graph showing body weight changes of mice
treated with adenoviru.s carrying a reporter gene (~-galactosidase), used
as a control.
Figure 4 i.s a graph .showing body weight changes of mice
S treated with adenovirus carrying the human leptin gene.
Figure 5 is a graph .showing the percent of body weight
changes for all groups of mice.
Figure 6A (left graph) is a graph showing the amount of
human leptin found in the plasma of mice treated with adenovirus
10 containing the leptin gene. ~igure 6B (right graph) shows re~sult.s for
mice treated with five daily injections of recombinant hum;m leptin.
Figure 7A (left graph) ~shows the amount of insulin and
leptin in plasma of mice treated with adenovirus containing the leptin
gene. Figure 7B (right graph) shows the amount of insulin and leptin in
15 plasma of mice treated with recombinant human leptin injections.
Figure X shows glucose levels of the mice treated with
either recombinant leptin, reporter gene or adenovirus containing the
leptin gene.
As u.sed throughout the specification and claims, the
following definitions apply:
"Native" a gene or protein is native if it naturally occurs in
a given organism.
"Leptin gene": a gene from any m~mm~l which encode.s a
native leptin, or a derivative thereof. A "derivative" is a modified
leptin molecule which retain~s at least ~0% of the biological activity of
native leptin.
"Leptin receptor": a gene from any mammal which encodes
a native leptin receptor, or a derivative thereof. A "derivative" is a
modified receptor molecule which binds native leptin at least ~0% as
efficiently as a native receptor molecule.
"Obesity regulating gene": a gene whose gene product is
involved in the regulation of obesity in a m~mm~l, including genes
encoding leptin, leptin receptors, neuropeptide Y, and the like.
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!~ ~
In the past, recombinant leptin ha,s been administered to
~nim~ls who exhibiting an obe,se phenotype, and a daily injection has
been shown to decrease body weight. There are numerous
S disadvantages to this method of treating obesity, however. First, this
method is helpful only for those ;lnim~l~s whose obesity is caused, at least
in part by an insufficient amount of leptin produced. Not all obesity is
due to this defect. Further, injections are not a particularly convenient
method of treatment, particularly for long-term treatments. In addition,
the half-life of leptin is short. so the duration of the treatment wa~s found
to be only about 24 hours, after which the animals were ob,served to re-
gain weight.
This invention solves the problems as~sociated with a daily
admini~stration of recombinant protein by providing a vector which can
express leptin or a leptin receptor in viv~. It has been surprisingly
found that leptin which is expressed in vivo is more advantageous than
administration of recombinant leptin; itls effects last longer, and most
surprisingly, is up to 20 fold more potent than recombinant leptin
a~lministered by injection.
Expression of a leptin receptor or neuropeptide Y in vivo
allows for treatment of heretofore untreatable types of obesity.
Genes
The sequences of leptin and leptin genes from various
species are known (Zhang et al., 1994 Nature 372:425; Ogawa et al.,
1995 J. Clin. Invcst. 96: 1647- 1652; Murakami ct al., 1995 Bioc~hem.
Biophys. Res. Commun. 209:944; and Con~sidine et (11., 1995
J. Clin. Invest. 95:29P~6; each of which is hereby incorporated by
reference). If desired, gene.s encoding leptin derivatives may also be
u.sed. Since the amino acid and nucleotide .sequence of leptin is known,
it is well within the .skill of one of the ordinary artisian to construct a
nucleotide sequence which encode~s a desired mutant form of leptin.
These can be used to study structure and function relationships involved
in leptin binding and signaling in the transgenic ~nim~l model.
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The amino acid .se4uence~s of leptin receptor,s and the
nucleic acid ,se~luence,s of genes encoding leptin receptors are also
known; see, for example Toriaglla et al., 1995, Cell ~3: 1-20 which is
hereby incorporated by reference. The leptin receptor,s can exist in
5 various isoform,s, due to alternate ,splicing. The biological consequence,s
of the presence of many of the,se isoforms i~ not clearly under,stood.
However, a mutation that results in premature termination of the long
form of the mouse leptin receptor i,s apparently responsible for the
obese phenotype of the (ll~ldh mouse (Lee ef al., 1996, Nature 379:632-
10 635; Chua et al., 1996, Seienee 271 :994-996; and Chen et al., 1996, Cell
~4:491 -495).
In further aspects of this invention, a derivative leptin
receptor is introduced into a m~mmal, and the resulting mammal can be
used to study structure and functional relationships between leptin
15 binding and the leptin receptor.
The gene which encodes the leptin or leptin receptor should
also contain at least one element which allows for expression of the gene
when introduced into the host cell environment. These se4uences
include, but are not limited to promoterls, response elements, and
20 enhancer elements. In a preferred aspect of thi.s invention, promoters
are chosen which are regulatable; i.e. are inducible rather than
constitutive. Particular ex~mples of such promoters include: Sr-alpha,
CMV, regulatable tet, P-450, albumin and the like.
25 Vector
The heterologous leptin or lepin receptor gene may be
delivered to the organism using a vector or other delivery vehicle.
DNA delivery vehicles can include viral vectors such as adenoviruses,
adeno-associated viruses, and retroviral vectors. See, for example: Chu
30 et al. 1994 Cene Ther 1: 292-299; Couture et al. 1994 ~um Gene Ther
5:667-677; and Eiverhand et al. 1995 Gene Ther 2: 336-343. Non-viral
vectors which are also suitable include DNA-lipid complexes, for
exarnple lipo~some-mediated or ligand/ poly-L-Lysine conJugates, such as
asialoglyco-protein-mediated delivery systems. See, for example:
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Felgner et al. 1994 J. Biol. Chem, 269: 2550-2561; Derossi et al. 1995,
Resto). Neurol. Neuros. ~: 7-10; and Abcallah et al. 1995 Biol. Cell ~5:
1 -7.
If a vector is chosen a.s the delivery vehicle for the obe.sity
5 regulating gene, it may be any vector which allows expres~sion of the
gene in the ho~t cells. It is preferable if the vector also i~ one which i.s
capable of integrating into the host genome, so that the gene can be
expressed permanently, but this is not required. In cases where the
vector does not integrate into the host genome, the expression of the
l0 gene may be transient rather than permanent.
One vector which is suitable for tran.sient expre~;sion of the
o~ gene i.s an adenovirus which has a deletion in the El gene. Such
vectors are known, as taught in the aforementioned WO 95/00655 and
Mitani et al., 1995 publications. The.se viruses preferentially infect
15 hepatocyte.s, where they persist for approximately 3-4 weeks after the
initial infection. While in the hepatocytes, these viruses can expres.s the
heterologous gene.
The vector is a~lmini~tered to the host, generally by IV
injection. Suitable titers will depend on a number of factors, such as the
20 particular vector chosen, the host, strength of promoter used and the
severity of the di~sease being treated. For mice, an adenovirus vector is
preferably administered as an injection at a dose range of from about
5.0 x 106 to about l0 x 106 plaque forming units (PFU) per gram body
weight. Preferred dosages range from at least about 6-9 x 106 PFU/gm
25 body weight, and more preferred is from at least about 6.7-~.6 x 106
PFU/gm body weight (e4uivalent to approximately at least 1-5 x 10
PFU for mice).
Thus this invention specifically is directed to a method of
treating obesity, elevated serum glucose levels or elevated insulin levels
30 which is, at least in part, due to an insufficient amount of leptin or leptin receptors by a m~mmal comprising:
a) transfecting the mammal with a viral vector
comprising a gene encoding leptin or leptin
receptors; wherein said vector further comprises at
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lea.st one element regulating its expression in a
mammalian ti.ssue; and
b) allowing sufficient time to pass for transcription and
tran~lation of the leptin gene.
s
Hosts
~ nim~ls which transiently express the ~b or o~7-receptor
gene products are valuable research tools. For example, they can be
used to monitor the effects of decreasing amounts of leptin, or the effect
10 of various exogenously supplied substances (such as hormones or
putative leptin receptor agonists and antagonists) in an environment of
decreasing leptin availability.
A.side from making animal models useful in studying
various aspects of obesity, this invention is specifically directed to gene
15 therapy for humans.
In accordance with this invention, mice which are obese
(oblob) have been injected with an adenovirus containing the human
leptin gene, although the leptin gene from any desired species may be
20 used, and in preferred embodiments. the gene which i.s from the same
species as the host is used. These were compared with obl~h mice
injected with an adenovirus containing only a marker gene (,~-
galactosidase), those receiving injections of recombinant leptin, and to
untreated control.s. Further controls used in some of the experiments
25 are ~lhldb mice (obese, but unrespon.sive to leptin injections).
Body-wei~ht: Figure 2 illustrates the body weight changes
for mice receiving 1 ~g/gm body weight human recombinant leptin
protein injections daily, compared to untreated controls. ~nim~l.s
30 receiving leptin were injected for five con~ecutive days, shown by the
darkened symbols on the graph. All the ~nim~ls receiving the leptin lost
weight within 24 hours post-injection. All ~nim~ls gain weight within
4~ hour.s after the last IP injection. Figure 3 illustrates the weight
measured for mice receiving variou.s titers of an adenovirus carrying
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the reporter gene. All ~nim;lls continued to gain weight post injection.
Figure 4 showls the result~s for mice receiving an adenoviru,s carrying
the leptin gene. As c;m be seen from the graph, all anirnal~s lo~st weight
within 24 hours post injection. They continued to loose weight for 1-2
5 weeks post treatment. The injection~s were effective over a relatively
large titer range, and a dose-effect was noted.
Along with absolute change~s in weight, the percentage body
weight change wa~s calculated for all group~s. In the animals treated with
recombinant leptin injections, weight los~s plateaued at day three, and
10 from day l-S post treatment, a 4.7% loss in body weight was noted. In
those mice treated with the vector carrying the leptin gene, wei~ht lo~ss
persisted over a 10-12 day period, and re~sulted in an 1~.61% lo~ss in
body weight. Furthermore, over day~s 1-5 post treatment, a 9.17% los~s
in body weight was ob~served compared to only 4.7% lo,ss in the
lS recombinant leptin treated mice. Thi,s is illustrated in Figure S.
Leptin: The amount of human leptin in plasma was
measured in the animals which received injections of human
recombinant leptin and those which received the vector carrying the
20 leptin gene. Tho,se receiving the recombinant protein were noted to
have leptin levehi which were approximately 20-fold higher than the
amount of leptin found in control (lean, wild type) animals; peak
amounts of 399.P~ + 40.91 ng/ml. Those receiving the leptin gene had
levels of leptin in their plasma which was within the normal range found
25 in a wild-type mouse (17.52 + 4.66 ng/ml). ln both groups of ~nim~l.s,
weight gain was synchronized with the fall of human leptin detected in
the plasma. This is illustrated in Figure 6.
In~sulin: The amount of insulin in the plasma was measured
30 in both the ~nimAls receiving recombinant protein and those which
received the gene therapy. This is illustrated in Figure 7. In both
groups, insulin levels were observed to drop to those found in lean
(wild-type) levels and was inversely correlated to leptin level.s. In the
mice receiving gene therapy, the low insulin levels were su.stained for at
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least one week whereas in the recombinant leptin-treated mice, insulin
levels increased to pre-treatment levels within 24 hours post injection.
Glucose: The levels of glucose in plasma was also
5 measured in mice receiving recombinant leptin and tho.se receiving the
gene therapy treatment. In both groups, the glucose level.s dropped
within 6-9 days post treatment. The recombinant protein-treated mice
did not achieve levels comparable to those found in lean, wild-type
mice, and only sustained the lower level for less than one week. On the
10 other hand, the mice which received the gene therapy had reductions in
glucose level,s to that of wild type lean mice, and they sustained this
reduced level for at least two weeks. This is illu.strated in Figure ~s.
The following non-limiting Example,s are presented to
better illustrate the invention.
EXAMPLE 1
Clonin and expression of leptin
Two PCR cDNA amplification fragments were obtained
20 from Jefferson University (generated by cloning both variants from ~
Clontech phage human hypothalamic library): one coding for the human
leptin and one for the human leptin variant with glutamine, (Zhang
e~ al., 1994, Nature 372:425; Considine e~ al., 1995 J. Clin. Invest.
95:29~6). Both PCR fragments were amplified for cloning purposes.
25 Two primers were designed and ordered from GIBCO BRL Custom
Primers: Forward primer: ATG CAT TGG GGA ACC CTG TG
Reverse primer: TCA GCA CCC AGG GCT GAG GT
The primer~s were used to re-amplify the cDNA as follows:
2 ,ul each primer (0.3 ,ug/~l stock), 2 ,ul dNTP (10 ~M, Pharmacia), 10
30 ~1 10 X PCR Buffer (Buffer 2 from Expand Long Template PCR
System Kit, Boehringer Mannheim), 2 ~I Taq polymerase (Perkin
Elmer), 3 ,ul template DNA and 1~ ~11 water. PCR cycling conditions
were as follows: Mixture was incubated at 94~C initially (without the
addition of the Taq enzyme) for 1-2 minute, Taq was then added to each
35 tube and the cycling program was initiated, 20 cycles of 94~C for 30
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11
seconds, 45~C for 45 second,s and 72~C for 1 minute. At the end of the
20 rounds of amplification the samples were incubated at 72~C for 7
minute.s. The expected fragment size in each case (human leptin - hOb,
and human leptin with glutamine-hObGLN) was 501 and 504,
5 respectively. The PCR amplified fragment,s were cloned into pCR-
Script SK(+) plasmid (Stratagene) and several selected bacterial
colonies were grown, plasmids extracted and sequenced to verify
correct sequence of both cloned.
Insert.s were then used for generating recombinant
10 adenoviral shuttle vector.s. The adenoviral vector.s used in this study are
essentially the sarne ~s those described in Morsey et al 1993 J. Cli~l.
Invest. 92: 15~0-~6, which is hereby incorporated by reference, except
for the leptin gene insert. The pdelE 1.sp 1 CMV-BGHpA adenoviral
shuttle vector, obtained from Baylor College of Medicine was used for
15 the cloning of the two inserts (hOb and hObGLN) Sirnilarly mOb
cDNA (Zhang et al., 1994 Nature 372:425) was inserted into
pdelE 1 sp I CMV-BGHpA. All three shuttles (pdelE 1 sp 1 CMV-mOb-
BGHpA~ pdelE 1 sp 1 CMV-hOb-BGHpA and pdelE 1 sp l CMV-hObGLN-
BGHpA') were tested for leptin expression by western blot analysi~s.
EXAMPLE 2
The three shuttle vectors from the previous example are
u.sed in rescue replication of the deficient El deleted adenoviral vectors.
25 293 cells, comrnercially available from Microbix, passage 27-30 were
set up one day ahead of transfection in 60mm dishels, and were about 70-
~0% confluent at the time of use.
Plate.s were made cont~ining one of the shuttle plasmids and
pJM17; pFG140 (purchased from Microbix Biosystems Inc.) was used as
30 a positive control for the efficiency of transfection. Plaques were
identified, and plugged out of the agarose overlay using a sterile glass
Pasteur pipette. E,ach plugged plaque was resuspended in 100-500 ~1 of
PBS (with calciurn and magnesium) in 10% glycerol, frozen at -~0~C
and thawed ( I -3 times). The thawed plaque was then used to infect a
35 90% confluent 6 cm plate of 293 cells to expand the isolated virus. 5-
~
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12
days post infection~ cytopathic effects (cpe) were apparent on the cells
(cells rounded up and started to detach and float in media). Cells were
collected by scraping and tested for leptin expression by western
analysis and for DNA restriction pattern by Hind III digestion of
5 extracted DNA and ethiduim bromide stained gel analysis. One of the
positive plaques identified based on leptin secretion and correct DNA
restriction pattern was .selected and used for a second plaque purification
followed by a similar procedure of expansion and analysis. After the
second plaque purification, the virus was propagated on a large scale.
10 Cesium banding and titration wa.s used to purify and quantitate.
The resulting titered viral stocks (Ad-HCMV-mOb-
BGHPA, Ad-HCMV-hOb-BGHPA and Ad-HCMV-hOb GLN-BGHPA)
were stored at -~0~C until use.
EXAMPLE 3
Tran,s~enlc mlce
Ba.seline determinations Three group,s of mice were u.sed~ loh, dhldb,
20 and lean (wild type, controls). All group.s of mice were fed milled
rodent chow (500~) ~starting from day of arrival or day after arrival.
Food consumption was also measured. After approximately 4 days on
milled chow, the mice were weighed and bled for determination of
plasma levels of glucose and insulin. Injections were started ~ days after
25 the initiation of base line measurements but before injections, mice were
weighed and blood samples were obtained from all study mice for
determination of plasma glucose and insulin. Leptin level in plasma
were also mea.sured.
Mice were hou~sed 5 per cage and fed milled Purina Chow
30 500~s in feed cups with lids. 24 hour food consumption was measured at
the same time each day. Only after food consumption was equilibrated
to a fairly constant level, usually 20-25 grams chow/5 mice-day, was
viru.s injected.
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~3
On the day of injection but before injection, food
consumption, body weight, and a ba.seline blood sample were taken in
the morning from a snipped end of tail. Blood wa.~ collected into
heparinized capillary tube (total volume approximately 70-100~1).
S Hematocrit was measured. and plasma wa,s collected.
Mice were injected as follows:
A. ohlol~ mouse ~roup,s: iv injections~ S mice/~roup
Group 1 received 2.75 x 10X / gm wt of AdHCMV-hob-BGHPA
(in 500111 dialysis buffer) in the tail vein.
Group 2 received 2.75 x 10~ / gm wt of AdHCMV-~3gal reporter
(in 500~1 dialysi~i buffer) in tail vein.
Group 3 received 500~1 dialysis buffer in tail vein.
Group 4 received I mg/kg wt active leptin daily ~P injections for
5 days.
B. ~lhldh mou~se ~roup,s: iv injection,s, 5 mice/~roup
Group 1 received 2.75 x 108 / gm wt of AdHCMV-hob-BGHPA
(in 500111 dialy,sis buffer) in the tail vein.
Group 2 received 2.75 x 10~ / gm wt of AdHCMV-~gal reporter
(in 500~1 dialysis buffer) in tail vein.
Group 3 received 1 mg/kg wt active leptin daily IP injections for
5 day,s.
C. Lean control mou,se group: one cage of 5 mice as measure of
lean parameter.s
No injections.
EXAMPLE 4
Leptin receptor
Adenovirus vectors are made similarly to those described in
Examples 2-3, except that the leptin receptor gene replaces the leptin
gene. Mice which are dhldh are u~sed in place of the ohloh mice.
Results for the di~Icl/7 mice are similar to those ob.served with the obloh
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14
mice reported herein. After injection, gluco.se levels fall, insulin levels
fall and the mice loose weight. No effect is observed in control mice
and in ohloh mice injected with vector carrying a leptin receptor gene.
S DETAILED DESCRIPTION OF THE INVENTION
Not Applicable
