CA2258504A1 - Gene therapy for obesity - Google Patents
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Abstract
Gene therapy can treat obesity in mammals. An obesity regulating gene is delivered to a mammal. Preferably, the gene encodes leptin or a leptin receptor. The protein which is delivered and expressed in vivo is more effective than protein which is injected into the animal.
Description
~ 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.
CA 022~8~04 1998-12-16 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.
. . . ~, CA 022~8~04 1998-12-16 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.
CA 022~8~04 1998-12-16 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.
CA 022~8~04 1998-12-16 !~ ~
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.
CA 022~8~04 1998-12-16 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:
CA 022~8~04 1998-12-16 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 CA 022~8~04 1998-12-16 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 CA 022~8~04 1998-12-16 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 CA 022~8~04 l998- l2- l6 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.
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 CA 022~8~04 1998-12-16 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.
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-~
CA 022~8~04 1998-12-16 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.
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.
CA 022~8~04 1998-12-16 ~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.
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 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
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.
CA 022~8~04 1998-12-16 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.
. . . ~, CA 022~8~04 1998-12-16 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.
CA 022~8~04 1998-12-16 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.
CA 022~8~04 1998-12-16 !~ ~
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.
CA 022~8~04 1998-12-16 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:
CA 022~8~04 1998-12-16 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 CA 022~8~04 1998-12-16 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 CA 022~8~04 1998-12-16 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 CA 022~8~04 l998- l2- l6 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.
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 CA 022~8~04 1998-12-16 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.
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-~
CA 022~8~04 1998-12-16 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.
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.
CA 022~8~04 1998-12-16 ~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.
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 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
Claims (13)
1. A method of treating obesity, lowering serum glucose levels or lowering serum insulin levels in a mammal in need of such therapy comprising delivering an obesity regulating gene to the mammal; wherein transcription and translation of the gene occurs in vivo.
2. A method of treating obesity, lowering serum glucose levels or lowering serum insulin levels in a mammal in need of such therapy comprising delivering a gene encoding a leptin to the mammal; wherein transcription and translation of the gene encoding leptin occurs in vivo.
3. A method according to Claim 2 wherein the heterologous gene encoding a leptin is delivered in a vector.
4. A method according to Claim 3 wherein the vector is an adenovirus.
5. A method according to Claim 4 wherein the adenovirus is expressed in liver.
6. A method according to Claim 1 wherein the mammal is a human.
7. A method of treating obesity which is, at least in part, due to an insufficient amount of leptin receptors produced by a mammal comprising:
a) transfecting the mammal with a viral vector comprising a gene encoding a leptin receptor;
wherein said vector further comprises at least one element regulating its expression in a mammalian tissue; and b) allowing sufficient time to pass for transcription and translation of the leptin receptor gene.
a) transfecting the mammal with a viral vector comprising a gene encoding a leptin receptor;
wherein said vector further comprises at least one element regulating its expression in a mammalian tissue; and b) allowing sufficient time to pass for transcription and translation of the leptin receptor gene.
8. A method according to Claim 7 wherein the vector is a viral vector.
9. A method according to Claim 8 wherein the vector is an adenovirus.
10. A method according to Claim 9 wherein the adenovirus is expressed in liver.
11. A method according to Claim 10 wherein the mammal is a human.
12. A vector comprising a gene encoding a leptin receptor; wherein said vector further comprises at least one element regulating its expression in a mammalian tissue.
13. A method according to Claim 7 wherein the vector is an adenovirus.
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| US60/020,812 | 1996-06-20 | ||
| GBGB9615787.0A GB9615787D0 (en) | 1996-07-26 | 1996-07-26 | Gene therapy for obesity |
| GB9615787.0 | 1996-07-26 |
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|---|---|
| CA2258504A1 true CA2258504A1 (en) | 1997-12-24 |
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| EP (1) | EP0921820A4 (en) |
| JP (1) | JP2000514422A (en) |
| CA (1) | CA2258504A1 (en) |
| WO (1) | WO1997048419A1 (en) |
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| US6620413B1 (en) | 1995-12-27 | 2003-09-16 | Genentech, Inc. | OB protein-polymer chimeras |
| US6541604B1 (en) | 1996-01-08 | 2003-04-01 | Genentech, Inc. | Leptin receptor having a WSX motif |
| US7074397B1 (en) | 1996-01-08 | 2006-07-11 | Genentech, Inc. | Method for enhancing proliferation or differentiation of a cell using ob protein |
| WO2000007014A2 (en) * | 1998-07-28 | 2000-02-10 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | Leptin-mediated gene-induction |
| US7208572B2 (en) | 1998-08-21 | 2007-04-24 | Albany Medical College | Leptin-related peptides |
| US6777388B1 (en) | 1998-08-21 | 2004-08-17 | Clf Medical Technology Acceleration Program, Inc. | Leptin-related peptides |
| US8227408B2 (en) | 2005-09-07 | 2012-07-24 | Neurotez, Inc. | Leptin as an anti-amyloidogenic biologic and methods for delaying the onset and reducing Alzheimer's disease-like pathology |
| KR20110028457A (en) | 2008-05-21 | 2011-03-18 | 뉴로테즈 인코포레이티드 | Methods for treating progressive cognitive disorders related to neurofibrillary tangles |
| CA2742600A1 (en) | 2008-11-04 | 2010-05-14 | Nikolaos Tezapsidis | Leptin compositions and methods for treating progressive cognitive function disorders resulting from accumulation of neurofibrillary tangles and amlyoid beta |
| LT3074033T (en) * | 2013-11-26 | 2019-02-25 | The Children`S Medical Center Corporation | Compounds for the treatment of obesity and methods of use thereof |
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| US6309853B1 (en) * | 1994-08-17 | 2001-10-30 | The Rockfeller University | Modulators of body weight, corresponding nucleic acids and proteins, and diagnostic and therapeutic uses thereof |
| JP2000507081A (en) * | 1995-05-08 | 2000-06-13 | カイロン コーポレイション | Nucleic acids for treating obesity |
| US6482927B1 (en) * | 1995-11-27 | 2002-11-19 | Millennium Pharmaceuticals, Inc. | Chimeric proteins comprising the extracellular domain of murine Ob receptor |
| ATE303437T1 (en) * | 1996-01-04 | 2005-09-15 | Amgen Inc | OB PROTEIN RECEPTOR AND RELATED COMPOSITIONS AND METHODS |
| EP0954579A1 (en) * | 1996-06-20 | 1999-11-10 | Merck & Co., Inc. | Gene therapy for obesity |
| US5869037A (en) * | 1996-06-26 | 1999-02-09 | Cornell Research Foundation, Inc. | Adenoviral-mediated gene transfer to adipocytes |
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1997
- 1997-06-20 WO PCT/US1997/012131 patent/WO1997048419A1/en not_active Application Discontinuation
- 1997-06-20 CA CA002258504A patent/CA2258504A1/en not_active Abandoned
- 1997-06-20 EP EP97932580A patent/EP0921820A4/en not_active Withdrawn
- 1997-06-20 JP JP10503629A patent/JP2000514422A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| EP0921820A1 (en) | 1999-06-16 |
| JP2000514422A (en) | 2000-10-31 |
| EP0921820A4 (en) | 2003-01-15 |
| WO1997048419A1 (en) | 1997-12-24 |
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