(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`1111111111111111 IIIIII IIIII IIII I 111111111111111 lllll lllll 11111111111111111111111
`
`(43) International Publication Date
`25 April 2002 (25.04.2002)
`
`PCT
`
`(10) International Publication Number
`WO 02/33063 Al
`
`(51) International Patent Classification 7: C12N 9/04, 15/53
`
`(21) International Application Number: PCT/KR0l/01271
`
`(22) International Filing Date:
`
`26 July 2001 (26.07.2001)
`
`[KR/KR]; 17-4, Daehyun I-dong, Puk-ku, Daegu 702-041
`(KR). CHOI, Myung-Sook [KR/KR]; #102-203 Gar(cid:173)
`denheights, Bumuh 4-dong, Soosung-ku, Daegu 706-014
`(KR). JUNG, Un-Ju [KR/KR]; #206, 112-8 Daehyun
`I-dong, Puk-ku, Daegu 702-041 (KR).
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`2000/61962
`
`20 October 2000 (20.10.2000) KR
`
`(71) Applicant (for all designated States except US): TG
`BIOTECH, INC. [KR/KR]; 452-2, Sungnae 1-dong,
`Kangdong-ku, Seoul 134-031 (KR).
`
`(74) Agent: LEE, Won-Hee; Sung-ji Heights II, 8th Floor,
`642-16 Yoksam-dong, Kangnam-ku, Seoul 135-080 (KR).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, lN, IS, JP, KE, KG, KP, KZ, LC, LK,
`LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX,
`MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL,
`TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.
`
`(71) Applicant and
`(72) Inventor: HUH, Tae-Lin [KR/KR]; #255-107 Dongsuh(cid:173)
`town Apt. Shinmae-dong, Soosung-ku, Daegu_706-781
`(KR).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): KOH, Ho-Jiu
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE,
`IT, LU, MC, NL, PT, SE, TR), OAPT patent (BF, BJ, CF,
`CG, CI, CM, GA, GN, GQ, GW, ML, MR, NE, SN, TD,
`TG).
`
`[Continued on next page}
`
`---iiiiiiiiiiii
`- -------------------------------------------
`iiiiiiiiiiii -
`!!!!!!!! -
`
`iiiiiiiiiiii
`
`(54) Title: ISOCITRATE DEHYDROGENASE, GENE THEREOF, AND USE OF THE SAME 1N THE TREATMENT OF OBE(cid:173)
`SITY, HYPERLIPIDEMIA, AND FATTY LIVER IN LIPID BIOSYNTHESIS
`
`iiiiiiiiiiii
`iiiiiiiiiiii
`
`120
`
`100
`
`...--..
`C
`>,.·-
`·- .......
`+-'
`(J)
`.> 0
`80
`+-'
`r..-
`0 Q.
`<( 0) 60
`0 E
`Q_ - -
`::J 40
`0
`
`E - 20
`
`■ Non-Tg
`111 Tg A
`
`~
`
`0
`
`\0 = ~
`Liver
`Adipocyte
`~ (57) Abstract: The present invention relates to a cytosolic isocitrate dehydrogenase, its gene, and its use in the treatment of obesity,
`~ hyperlipidemia, and fatty liver. The expression of the ID Pc gene and the concomitant increase in ID Pc level bring about an increase
`in the cellular level of NADPH, which causes the lipid deposition in adipocytes, leading to obesity and fatty liver. A decrease in
`0 the cellular level of NADPH, resulting from the suppression of the gene expression of IDPc, has the effect of inhibiting the lipid
`> deposition in adipocytes. Further, by taking advantage of the suppressive or inhibitory effects of isocitrate dehydrogenase inhibitors,
`
`__.., pharmaceutically effective materials for the prophylaxis and treatment of obesity, hyperlipidemia and fatty liver can be developed.
`
`Rigel Exhibit 1033
`Page 1 of 99
`
`

`

`WO 02/33063 Al
`
`1111111111111111 IIIIII IIIII IIII I 111111111111111 lllll lllll 11111111111111111111111
`
`Published:
`with international search report
`entirely in electronic form (except for this front page) and
`available upon request from the International Bureau
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`Rigel Exhibit 1033
`Page 2 of 99
`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`ISOCITRATE DEHYDROGENASE, GENE THEREOF, AND USE OF
`
`THE SAME IN THE TREATMENT OF OBESITY.
`
`HYPERLIPIDEMIA. AND FATTY LIVER IN LIPID
`
`BIOSYNTHESIS
`
`5
`
`FIELD OF THE INVENTION
`
`The present invention relates to an isoci trate
`
`dehydrogenase which ~- v~.~
`
`the production of NADPH
`
`10
`
`necessary for the biosynthesis of lipids,
`
`including
`
`fatty acids, squalene and cholesterol, and its use in
`
`the treatment of metabolic diseases, including obesity,
`
`hyperlipidemia and fatty liver. Also,
`
`the present
`
`invention relates to an isocitrate dehydrogenase gene,
`
`15
`
`fused
`
`gene
`
`constructs
`
`containing
`
`the
`
`gene,
`
`transfectant cells harboring the genes in their genome,
`
`and
`
`transgenic
`
`animals
`
`capable
`
`of
`
`expressing
`
`isocitrate dehydrogenase continuously throughout their
`
`20
`
`BACKGROUND OF THE INVENTION
`
`Taking part in the TCA (tricarboxylic acid) cycle,
`
`isocitrate
`
`dehydrogenase
`
`the
`
`oxidative
`
`25
`
`decarboxylation of citric acid into a-ketoglutarate
`
`with concurrent production of NADH or NADPH.
`
`In higher
`
`animals,
`
`isocitrate
`
`dehydrogenase
`
`SUBSTITUTE SHEET (RULE 26)
`
`Rigel Exhibit 1033
`Page 3 of 99
`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`isozymes can be separated into three classes according
`
`to
`
`their cofactors
`
`and
`
`locations
`
`in
`
`the cell:
`
`mitochondrial NAD+-dependent
`
`isocitrate dehydrogenase
`
`referred
`
`to as
`
`"IDH"), mitochondrial
`
`5 NADP+-dependent
`
`isocitrate dehydrogenase
`
`(hereinafter
`
`referred to as "IDPm"), and cytoplasmic NADP+-dependent
`
`isocitrate dehydrogenase (hereinafter referred to as
`
`"IDPc") . Among
`
`these isoci tr ate isoenzymes,
`
`IDH has
`
`been assumed to play a major role in the oxidative
`
`10
`
`decarboxylation of
`
`isocitrate
`
`in
`
`the
`
`tricarboxylic
`
`acid cycle
`
`(TCA) with concurrent production of a(cid:173)
`
`ketoglutarate and NADH.
`
`NADH
`
`is used for energy
`
`generation through the electron transfer system and a(cid:173)
`
`ketoglutarate is a metabolite used in the synthesis of
`
`15
`
`amino acids such as glutamic acid, glutamine, arginine,
`
`and praline,
`
`and other biological products.
`
`IDH
`
`activity is regulated as a control point of the TCA
`
`cycle. Therefore, IDH is a key enzyme to regulate not
`
`only
`
`the TCA cycle, but also energy metabolism,
`
`20 protein biosynthesis and nitrogen metabolism because
`
`metabolites of
`
`the TCA cycle
`
`take part
`
`in
`
`such
`
`metabolisms.
`
`Since its isolation from yeast and pig,
`
`IDH has
`
`been under study.
`
`Yeast
`
`IDH
`
`is an allosterically
`
`25
`
`regulated enzyme that exists as an octamer composed of
`
`two nonidentical subunits IDHl and IDH2 sharing high
`
`homology with each other.
`
`I DHl plays a role in the
`
`2
`
`Rigel Exhibit 1033
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`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`regulation of
`
`the
`
`enzyme activity while
`
`IDH2
`
`is
`
`responsible for the catalytic activity (Keys, D. A. &
`
`McAlister-Henn, L.,
`
`J. Bacterial., 172,
`
`4280-4287,
`(a, p,
`subunits), swine IDH also exists as an octamer (2(a 2p
`
`1990).
`
`5
`
`Broken down
`
`into
`
`three subunits
`
`y
`
`y)) in active form.
`
`Found to have bipartite structures, IDPm and IDPc
`
`are,
`
`however,
`
`not
`
`known
`
`as
`
`to
`
`their
`
`functions.
`
`Although both having molecular weight of about 45 kDa
`
`10 with high homology, the two enzymes were identified as
`
`different,
`
`independent proteins,
`
`as
`
`analyzed
`
`by
`
`immunological
`
`reaction experiments using polyclonal
`
`antibodies (Plaut, G. W. E. et al., Biochem. Biophys.
`
`Acta., 760, 300-308, 1983; Fantania, H. R. et al.,
`
`15
`
`FEBS, 322, 245-248, 1993).
`
`Particularly,
`
`IDPm and
`
`IDPc are highly tissue-specific.
`
`In cardiac muscle
`
`tissues, for instance, more than 90 % of total NADP+-
`
`dependent
`
`isocitrate
`
`dehydrogenase
`
`exists
`
`mitochondria and
`
`the
`
`remaining 10 % is
`
`found
`
`in
`
`in
`
`20
`
`cytoplasm.
`
`In contrast, it is reported that as low as
`
`3
`
`9,-
`0
`
`of
`
`the
`
`total
`
`NADP+ -dependent
`
`dehydrogenase
`
`of
`
`liver
`
`tissues
`
`is
`
`found
`
`in
`
`mitochondria while
`
`the
`
`remaining
`
`97
`
`g,.
`0
`
`exists
`
`in
`
`cytoplasm (Plaut, G. W. E., Current Topics
`
`in Cell
`
`25 Regulation, 2, 1-27, 1983).
`
`As mentioned
`
`above,
`
`isocitrate dehydrogenase
`
`isozymes have been characterized concerning some of
`
`3
`
`Rigel Exhibit 1033
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`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`their structural characteristics, but not concerning
`
`functions.
`
`~~,~~~-~~
`
`nowhere had been
`
`found
`
`studies on precise mechanisms of IDPm and IDPc until
`
`the publication of recent reports which merely made
`
`5
`
`the assumption that IDPm catalyzes a reverse reaction
`
`in the TCA cycle to convert a-ketoglutarate through
`
`isocitrate to citrate, which
`
`is associated with a
`
`tricarboxylate
`
`carrier
`
`to
`
`supply
`
`acetyl-CoA,
`
`a
`
`precursor for
`
`the biosynthesis of fatty acids and
`
`10
`
`cholesterol, with concurrent conversion of the citrate
`
`to
`
`oxaloacetate
`
`to
`
`raise
`
`cytoplasmic
`
`phosphoenolpyruvate
`
`levels,
`
`thereby
`
`promoting
`
`gluconeogenesis
`
`(Des Rosiers, C. et al., J. Biol.
`
`Chem., 269, 27179-27182, 1994; Fernandez, C. A. et al.,
`
`15
`
`J. Biol. Chem., 270, 10037-10042, 1995).
`
`Significance in gluconeogenesis is suggested for
`
`IDPm owing to its catalysis of a reverse reaction of
`
`the TCA cycle.
`
`In contrast, none of the reports for
`
`IDPc are concerned with its metabolic functions.
`
`IDPc
`
`20
`
`is known to be expressed in large quantities in the
`
`ovary and the mammary gland. Of the NADPH producing
`
`enzymes
`
`existing
`
`in
`
`rat
`
`liver,
`
`IDPc
`
`has
`
`been
`
`quantitatively analyzed to produce NADPH
`
`in greater
`
`quantities than do
`
`important enzymes of the pentose
`
`25
`
`phosphate
`
`pathway;
`
`i.e.,
`
`glucose-6-phosphate
`
`dehydrogenase
`
`for
`
`the
`
`conversion
`
`of
`
`glucose-6-
`
`phosphate to 6-phosphoglucono-8-lactone and NADPH, 6-
`
`4
`
`Rigel Exhibit 1033
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`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`phosphogluconate dehydrogenase for the conversion of
`
`6-phosphogluconate to ribulose-5-phosphate and NADPH,
`
`and cytoplasmic malic enzyme for
`
`the conversion of
`
`malate to pyruvate and NADPH; by factors of 16, 8 and
`
`5
`
`18, respectively
`
`(Veech, R. L. et al., Biochem. J.,
`
`115, 609-619, 1969).
`
`In
`
`cytoplasm,
`
`various
`
`enzymes
`
`involved
`
`in
`
`metabolisms of fatty
`
`acids,
`
`cholesterol and hormones
`
`a large quantity of NADPH for their
`
`10 activities. Thus far, the NADP producing enzymes such
`
`as glucose-6-phosphate dehydrogenase and malic enzyme
`
`have been believed
`
`to play an
`
`important
`
`role
`
`in
`
`?upplying NADPH to cytoplasm. However, in light of its
`
`ability to produce cytoplasmic NADPH,
`
`IDPc is expected
`
`15
`
`to be more
`
`responsible
`
`for
`
`the regulation of
`
`the
`
`supply of NADPH. Ultimately, it is assumed that IDPc
`
`plays a crucial role in the biosynthesis of fatty
`
`acids and cholesterol. Among the fatty acid synthases
`
`in
`
`the biosynthesis of
`
`acids, ~-
`
`20
`
`ketoacyl-ACP reductase and enoyl-ACP reductase require
`
`NADPH as a cofactor for
`
`their catalysis.
`
`In
`
`the
`
`biosynthesis of cholesterol, a large quantity of NADPH
`
`is required for
`
`the reactions catalyzed by HMG-CoA
`
`reductase and squalene
`
`and for the final
`
`25
`
`19-step
`
`reaction
`
`from
`
`lanosterol
`
`to cholesterol.
`
`Accordingly, control of the activity of IDPc, which
`
`functions to supply most of the NADPH required in the
`
`5
`
`Rigel Exhibit 1033
`Page 7 of 99
`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`cell, is very important to regulate the biosynthesis
`
`of fatty acids and their derivatives, lipids,
`
`and cholesterol and its derivatives.
`
`In higher animals, lipid deposition follows the
`
`5
`
`fallowing procedure. When excess energy sources are
`
`available,
`
`the differentiation of adipose cells is
`
`resulting in an increase in the number
`
`and size of white adipose
`
`tissues with concomitant
`
`deposition of lipids.
`
`In
`
`turn,
`
`the white adipose
`
`10
`
`tissue allows the ob gene to be actively expressed,
`
`which leads to an increase in body leptin level.
`
`In
`
`response, the hormonal action in the brain is changed
`
`toward the decreasing of appetite. Meanwhile, excess
`
`calories are consumed to maintain the body temperature,
`
`15
`
`using uncoupler proteins
`
`(UCP) .
`
`In the white adipose
`
`tissue, expression of the genes which encode master
`
`transcription factors for the proliferation of adipose
`
`cells,
`
`such
`
`as
`
`peroxysome proliferator-activated
`
`receptor
`
`y
`
`(PPARy),
`
`C/EBPa
`
`and ADDl/SREBPl,
`
`is
`
`20
`
`activated.
`
`Thus, adipose cell differentiation and
`
`lipid deposition are promoted and excess body energy
`
`is stored
`
`in lipid
`
`form,
`
`so
`
`that body energy is
`
`balanced (Hu, E. et al., Proc. Natl. Acad. Sci. USA,
`
`92, 9856-9860, 1995; Keller, H. et al., Proc. Natl.
`
`25 Acad. Sci. USA, 20, 9856-9860, 1993; Freytag S. O., et
`
`al., Genes Dev., 8, 1654-1663, 1994; Tontonoz, P. et
`
`al., Mol. Cell. Biol., 13, 4753-4759, 1993; Spiegelman,
`
`6
`
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`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`B. M., Cell, 87, 377-389, 1996). Examples of ligands
`
`necessary
`
`for
`
`the activation of PPARy,
`
`a master
`
`transcription factor for adipose cell differentiation,
`
`include polyunsaturated fatty acids such as linoleic
`
`5
`
`acid, docosahexanoic acid (DHA), and arachidonic acid
`
`(Krey, G. et al., Mol. Endocrinol., 11, 779-791, 1997;
`
`Yu et al., J. Biol. Chem., 270, 2397 5-23983, 1995) .
`
`Also, prostaglandin J2 is known to serve as a ligand
`
`of the master
`
`ranscri
`
`factor
`
`( Forman B. M. et
`
`10 al., Cell, 83, 803-812, 1995; Kliewer S. A. et al.,
`
`Cell, 83, 813-819, 1995).
`
`From
`
`this
`
`review,
`
`there
`
`is concluded a high
`
`possibility that IDPc might be directly involved in
`
`controlling the biosynthesis of various
`
`acids,
`
`15
`
`cholesterol and hormones owing
`
`to
`
`its ability
`
`to
`
`produce NADPH. Also, IDPc can be assumed to play a key
`
`role in obesity and fatty liver by encouraging the
`
`production of activating ligands for PPARy,
`
`such as
`
`polyunsaturated
`
`acids and arachidonic acid to
`
`20
`
`trigger
`
`the
`
`cascade expression of various genes
`
`related
`
`to
`
`the differentiation of adipose cells.
`
`Additionally,
`
`the requirement of a
`
`large quantity of
`
`NADPH
`
`for
`
`cholesterol
`
`biosynthesis
`
`offers
`
`the
`
`possibility that artificial control of intracellular
`
`25
`
`levels of ID Pc and its reaction product NADPH might
`
`provide
`
`a
`
`means
`
`of
`
`controlling
`
`cholesterol
`
`biosynthesis.
`
`7
`
`Rigel Exhibit 1033
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`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`SUMMARY OF THE INVENTION
`
`Leading to the present invention, the intensive
`
`5
`
`and
`
`thorough
`
`research on
`
`the mechanism of
`
`lipid
`
`biosynthesis
`
`through molecular
`
`biological
`
`and
`
`biochemical
`
`experiments using
`
`transfectant
`
`animal
`
`cells and transgenic mice, conducted by the present
`
`inventors, resulted in the finding that intracellular
`
`10
`
`levels of IDPc and its reaction product NADPH have a
`
`decisive
`
`influence on not only
`
`the differentiation
`
`rate of adipose cells and lipid deposition in adipose
`
`cells, but the biosynthesis of lipids and cholesterol.
`
`Therefore,
`
`it is an object of
`
`the present
`
`15
`
`invention
`
`to provide
`
`an
`
`isoci trate dehydrogenase
`
`enzyme for producing NADPH, and its gene.
`
`It is another object of the present invention to
`
`provide a fused gene construct which contains a gene
`
`encoding isocitrate dehydrogenase, a transfectant cell
`
`20 which harbors the gene in its genome, and a transgenic
`
`animal which
`
`can
`
`express
`
`the
`
`gene
`
`continuously
`
`throughout its lifespan.
`
`It is a further object of the present invention
`
`to provide the use of ~~v~•~
`
`dehydrogenase and its
`
`25
`
`gene
`
`in
`
`the
`
`treatment and prophylaxis of obesity,
`
`hyperlipidemia, and fatty liver or in the biosynthesis
`
`of lipids.
`
`8
`
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`

`WO 02/33063
`
`PCT/KR0l/01271
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`
`Fig.
`
`1
`
`provides
`
`schematic
`
`diagrams
`
`showing
`
`5
`
`structures of a basic LNCX-vector (top), a recombinant
`
`vector into which an
`
`IDPc gene is inserted in the
`
`sense orientation to increase the expression of the
`
`IDPc gene in NIH3T3 F442A adipocytes (middle), and a
`
`recombinant vector into which an IDPc gene is inserted
`
`10
`
`in
`
`the
`
`antisense
`
`orientation
`
`to
`
`decrease
`
`the
`
`expression of the IDPc gene in NIH3T3 F442A adipocytes
`
`(bottom).
`
`Fig. 2a provides optical photographs showing Oil(cid:173)
`
`Red-0-dyed
`
`adipocytes differentiated
`
`from
`
`normal
`
`15 NIH3T3 F442A (left),
`
`the transfectant FSl cells with
`
`improved
`
`IDPc
`
`gene
`
`expression
`
`(middle),
`
`and
`
`the
`
`transfectant FASl cells with decreased
`
`IDPc gene
`
`expression (right) on plates (upper panel) and in part,
`
`magnified at 200 power (lower panel).
`
`20
`
`Fig. 2b provides optical photographs showing the
`
`lipid deposition in adipocytes, which is in a NADPH
`
`dose-dependent pattern.
`
`Fig. 3 is a diagram illustrating the construction
`
`of
`
`a
`
`recombinant
`
`expression vector
`
`for use
`
`in
`
`25
`
`generating a transgenic animal, in which an IDPc cDNA
`
`is
`
`inserted
`
`downstream of
`
`a
`
`rat-derived
`
`PEPCK
`
`(phosphoenolpyruvate carboxykinase) gene promoter.
`
`9
`
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`PCT/KR0l/01271
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`Fig. 4 provides photographs showing a comparison
`
`in body size and epididymal fat pad deposit between F1
`progeny
`from
`the
`transgenic mice of
`the present
`
`invention and normal mice.
`
`5
`
`Fig.
`
`5 provides
`
`autoradiographs
`
`showing
`
`an
`
`increase in the expression level of obesity-indicative
`
`genes in the adipose tissue of the transgenic mice of
`
`the presen.t invention, compared to normal mice.
`
`Fig. 6a is a histogram comparing the body weight
`
`10
`
`of the transgenic mice F1 to that of normal mice.
`Fig. 6b is a histogram comparing the liver weight
`
`of the transgenic mice F1 to that of normal mice.
`6c
`is a histogram comparing
`
`the
`
`IDPc
`
`activity and blood IDPc level of the transgenic mice F1
`to those of normal mice.
`
`15
`
`Fig.
`
`6d
`
`is
`
`a
`
`histogram
`
`comparing
`
`the
`
`[NADPH]/[NADPH+NADP+] of the transgenic mice F1 to that
`of normal mice.
`
`Fig. 6e is a histogram comparing the epididymal
`
`20
`
`fat pad weight of the
`
`._.,_c:a.u.::,,.p::;u.,. mice F1 to that of
`
`normal mice.
`
`Fig. 6f
`
`is a histogram comparing
`
`the blood
`
`triglyceride and cholesterol levels of the transgenic
`
`mice F1 to those of normal mice.
`Fig. 6g is·a histogram comparing the triglyQeride
`
`25
`
`and cholesterol levels in the liver of the transgenic
`
`mice F1 to those of normal mice.
`
`10
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`Fig. 6h is a histogram comparing the blood leptin
`
`level of the transgenic mice
`
`to that of normal mice.
`
`Fig.
`
`7a provides photographs
`
`showing
`
`liver
`
`tissues of
`
`the
`
`transgenic mice of
`
`the present
`
`5
`
`invention and the control mice.
`
`Fig. 7b provides photographs showing adipocyte of
`
`the transgenic mice of the present invention and the
`
`control mice.
`
`Fig. Sa is a graph illustrating the inhibitory
`
`10
`
`activity of
`
`oxalomalic
`
`acid
`
`against
`
`isocitrate
`
`dehydrogenase activity.
`
`Fig. Sb is a graph illustrating the inhibitory
`
`activity of methyl
`
`isoci tr ate against
`
`isoci trate
`
`dehydrogenase activity.
`
`15
`
`Fig. 9 provides optical photographs showing Oil-
`
`Red-O-dyed adipocytes differentiated from NIH3T3 F442A
`
`cell
`
`treated with
`
`no
`
`isocitrate
`
`dehydrogenase
`
`inhibitors
`
`(left), oxalomalate
`
`(middle), and methyl
`
`isocitrate
`
`), magnified at 100 power
`
`(upper
`
`20
`
`panel) and 200 power (lower panel).
`
`Fig. 10a is a histogram illustrating comparing
`
`the weights of the liver and epididymal fat pad of the
`
`rats in which the isoci tr ate dehydrogenase inhibitor
`
`of the present invention is administered, to those of
`
`25
`
`rats administered with no inhibitors.
`
`Fig. 10b is a histogram illustrating comparing
`
`the blood triglyceride and cholesterol levels of the
`
`11
`
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`

`

`WO 02/33063
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`
`rats into which the isocitrate dehydrogenase inhibitor
`
`of the nr-oQ,or,r invention is administered, to those of
`
`non-administered rats.
`
`Fig. 10c is a histogram illustrating comparing
`
`5
`
`the blood HDL
`
`level of
`
`the rats
`
`into which
`
`the
`
`isocitrate dehydrogenase
`
`inhibitor of
`
`the present
`
`invention is administered, to that of non-administered
`
`rats.
`
`10
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`In an aspect, the present invention pertains to
`
`an isocitrate dehydrogenase enzyme which catalyzes the
`
`production of NADPH necessary for the biosynthesis of
`
`15
`
`fatty acids and cholesterol and
`
`the deposition of
`
`lipids,
`
`and
`
`to
`
`a
`
`gene
`
`encoding
`
`the
`
`isoci trate
`
`dehydrogenase.
`
`Useful
`
`in
`
`the present
`
`invention is
`
`the
`
`IDPc
`
`isolated from mice. The mouse-derived IDPc gene of the
`
`20
`
`present invention, as listed in Sequence No. 3, has an
`
`open reading frame
`
`(ORF) 1,245 bp in size, with a 3'(cid:173)
`
`untranslated region
`
`(UTR)
`
`in which a base sequence
`
`AAT'AAA,
`
`a putative poly-A signal, exists.
`
`The
`
`IDPc
`
`protein for which the IDPc gene codes consists of 414
`
`25
`
`amino acids,
`
`listed
`
`in Sequence No.
`
`4, with
`
`a
`
`molecular weight of 46,575 Da. Alignment of the IDPc
`
`amino acid sequences
`
`from various species
`
`indicates
`
`12
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`

`

`WO 02/33063
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`
`that the mouse IDPc of the present invention shares a
`
`homology of 97.8 % with rat IDPc, 68.5 % with bovine
`
`IDPm, and 64. 4 % with yeast IDPc. Particularly, an
`
`amino acid sequence from 412 to 414 of the mouse IDPc
`
`5
`
`is identical to the target sequence of peroxisome,
`
`which is known to be involved in the biosynthesis and
`
`degradation of fatty acids and cholesterol. Therefore,
`
`this suggests the high possibility that IDPc moves to
`
`peroxisomes and takes part in the synthesis of
`
`10
`
`acids and cholesterol thereat.
`
`In addition to IDPc,
`
`IDPm, a gene having a base
`
`sequence similar to that of the IDPc gene,
`
`is also
`
`used for producing NADPH required for the biosynthesis
`
`of
`
`acids and cholesterol and the deposition of
`
`15
`
`lipids in accordance with the present invention.
`
`In another aspect, the present invention pertains
`
`to a fused gene construct containing the gene, a novel
`
`cell strain which anchors the gene, and a transgenic
`
`animal which
`
`expresses
`
`the
`
`gene
`
`continuously
`
`20
`
`throughout its lifespan.
`
`To
`
`this end,
`
`first,
`
`the gene of
`
`interest is
`
`inserted into a mammalian expression vector in such a
`
`way as to transcribe the gene in the sense direction
`
`or in the antisense direction.
`
`25
`
`In this regard, retroviral expression vectors are
`
`preferably used as
`
`the gene carrier, with highest
`
`preference for pLNCX retroviral vector.
`
`pLNCX, which
`
`13
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`

`WO 02/33063
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`
`is derived from MMLV
`
`(Moloney murine leukemia virus),
`
`has a CMV
`
`( cytomegalovirus) promoter for expressing
`
`exogenous genes
`
`in mammalian cells, and a neomycin
`
`gene as a
`
`cn:;;;..1.1:;;;•1..,
`
`marker, along with an LTR
`
`( long
`
`5
`
`terminal repeat) sequence, an identification factor of
`
`retroviral vectors.
`
`Among
`
`the fused gene constructs thus prepared,
`
`those which are transcribed in the sense direction by
`
`the CMV promoter are used to enhance the chµLc~~..1.u of
`
`10
`
`IDPc, while those designed for antisense transcription
`
`are used
`
`to
`
`suppress
`
`the expression.
`
`Resultant
`
`recombinant vectors are
`
`introduced
`
`into NIH3T3 Ll
`
`cells,
`
`a
`
`kind of preadipocytes.
`
`Of
`
`the cell
`
`transfectants which were identified to have integrated
`
`15
`
`the IDPc gene into their genomes, the cells in which
`
`IDPc gene were inserted in the sense direction were
`
`named FSl, which was deposited with
`
`the Korean
`
`Collection
`
`for Type Culture
`
`of Korea Research
`
`Institute of Bioscience and Biotechnology
`
`(KRIBB)
`
`20
`
`under the deposition No. KCTC 0861BP, on Sep. 6, 2000.
`
`On the other hand, cell strains which have the IDPc
`
`gene inserted in the antisense direction were named
`
`FASl.
`
`Compared to the mouse NIH3T3 Ll cells into which
`
`25
`
`only the pLNCX vector was
`
`introduced (control) ,
`
`the
`
`enzyme activity was measured to be higher by about 2
`
`fold in the mouse NIH3T3 11 transfectant cell in which
`
`14
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`
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`
`the
`
`IDPc gene was
`
`inserted· in the sense direction
`
`(FSl), but lower by about 0.4 fold in the mouse NIH3T3
`
`Ll
`
`transfectant cells
`
`in which
`
`the
`
`IDPc gene was
`
`inserted in the antisense direction (FASl).
`
`5
`
`The effect of IDPc on the biosynthesis of fatty
`
`acids can be quantitatively measured with Oil-Red-0, a
`
`dye specific for
`
`lipids, which
`
`is applied
`
`to
`
`the
`
`adipocytes which have been differentiated
`
`from
`
`the
`
`transfectant cells after treatment with insulin. As a
`
`10
`
`result,
`
`lipid production was
`
`found
`
`to be conducted
`
`more actively
`
`in
`
`the
`
`transfectant cell FSl with
`
`improved IDPc gene expression than the control cell.
`
`On
`
`the other hand,
`
`little deposition of lipids was
`
`found in the transfectant cells FASl with lowered IDPc
`
`15
`
`gene expression, compared to the control cell (see Fig.
`
`2a). While being differentiated to adipocytes in the
`
`presence of NADPH, which
`
`an enzymatic reaction
`
`product of IDPc,
`
`the transfectant cells into which
`
`only pLNCX was
`
`introduced,
`
`that is, control cells,
`
`20
`
`show
`
`higher
`
`differentiation
`
`rates
`
`and
`
`larger
`
`intracellular lipid deposits as the concentration of
`
`NADPH increases (see Fig. 2b). These results indicate
`
`that IDPc, its gene, or its enzymatic reaction product
`
`NADPH plays a key role in determining intracellular
`
`25
`
`lipid deposits.
`
`Next,
`
`in order
`
`to examine
`
`the activity of
`
`isocitrate dehydrogenase, the fused gene construct is
`
`15
`
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`Page 17 of 99
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`

`WO 02/33063
`
`PCT/KR0l/01271
`
`used to prepare a transgenic animal which harbors the
`
`IDPc gene within its genome.
`
`1. Preparation of Fused Gene Construct
`
`5
`
`To express the IDPc gene permanently,
`
`there is
`
`required the integration of the gene into the genome
`
`of an animal. To this end, first, it is necessary to
`
`construct a recombinant vector which can express the
`
`gene of interest in mammals.
`
`In the resulting fused
`
`10
`
`gene construct, the expression of the gene of interest
`
`is regulated under a suitable promoter gene. The term
`
`~fused gene construct" as used herein, means
`
`a
`
`functional assembly of genes for use in transformation
`
`of certain organisms, which is comprised essentially
`
`15
`
`of at least one structural gene, and at least one cis(cid:173)
`
`acting
`
`regulatory
`
`element
`
`for
`
`controlling
`
`the
`
`expression of the structural gene.
`
`Generally, a cis-acting regulatory element may be
`
`in the form of a promoter, an enhancer, an intron, a
`
`20
`
`5'-UTR (untranslated region), and a 3'-UTR.
`
`In a fused
`
`gene construct, the cis-acting regulatory element may
`
`be located at any site of 10 kb or less distant from
`
`the 5'-flanking region, 3'-flanking region, 5'-end or
`
`3'-end of the structural gene or inside the structural
`
`25
`
`gene (in the case of an intron) .
`
`In addition to the
`
`structural gene and cis-acting regulatory element, the
`
`fused
`
`gene
`
`construct
`
`further
`
`comprises
`
`various
`
`16
`
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`Page 18 of 99
`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`components,
`
`including a polyadenylation signal
`
`for
`
`improving
`
`transcription or
`
`translation
`
`rates,
`
`a
`
`ribosome-binding sequence, an intron, etc. Further to
`
`these, a base sequence for improving the efficiency of
`
`5
`
`the insertion of a gene of interest into the genome or
`
`certain sites, and a marker gene for identifying the
`
`insertion may be provided for the fused gene construct.
`
`A promoter for the fused gene construct to be
`
`used in making a
`
`transgenic animal
`
`include the CMV
`
`10
`
`promoter, or expression regulatory regions for genes
`
`expressible in white adipose tissues, such as genes
`
`coding
`
`for
`
`lipoprotein
`
`lipase
`
`(LPL),
`
`adipsin,
`
`adipocyte protein 2
`
`(aP2) and IDPc.
`
`In a preferred
`
`embodiment of the present invention, there is employed
`
`15
`
`a
`
`rat-derived
`
`promoter
`
`for
`
`a
`
`cytosolic
`
`phosphoenolpyruvate carboxykinase
`
`(PEPCK) gene, which
`
`is expressed in both the liver and the white adipose
`
`tissues.
`
`In more detail, the preparation of a transgenic
`
`20
`
`animal in which the permanent expression of the IDPc
`
`gene is conducted starts with the cytosolic PEPCK gene
`
`of rats. From this gene, a 2.2 kb 5'-upstream sequence
`
`containing a promoter was obtained. Downstream of this
`
`sequence, a mouse IDPc cDNA was inserted in the sense
`
`25 orientation to prepare a fused gene construct, which
`
`was named pPEPCKIDPc.
`
`There are two kinds of PEPCK
`
`genes: one codes for a cytosolic enzyme and the other
`
`17
`
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`
`

`

`WO 02/33063
`
`PCT/KR0l/01271
`
`for a mitochondrial enzyme.
`
`In the present invention,
`
`a 5'-upstream
`
`sequence of
`
`the gene encoding
`
`the
`
`cytosolic PEPCK (hereinafter referred to as "PEPCK-C")
`
`was employed.
`
`In the liver,
`
`the intestine and the
`
`5
`
`kidney,
`
`the
`
`tissues, where
`
`the PEPCK-C
`
`gene
`
`is
`
`expressed under the regulation of the promoter, are
`
`determined
`
`depending
`
`on
`
`the
`
`regulatory
`
`regions
`
`existing in the 5'-upstream sequence. The 2.2 kb 5'(cid:173)
`
`upstream sequence of the PEPCK-C gene used in
`
`the
`
`10 present invention contains a gene sequence near nt 987,
`
`which is known as a regulatory region necessary for
`
`efficient expression in white adipose
`
`(Hanson,
`
`R. W. Annu. Tev. Biochem., 66, 581-611, 1997).
`
`Mice are useful for making
`
`ransae:ni
`
`animals,
`
`15
`
`but any animal,
`
`if it can be made
`
`transgenic,
`
`is
`
`available in the present invention because IDPc is an
`
`enzyme expressed in all higher animals.
`
`2. Preparation of Embr_yo
`
`20
`
`One of the most important steps in making of a
`
`transgenic animal
`
`is
`
`to
`
`introduce
`
`the
`
`fused gene
`
`construct
`
`into
`
`an
`
`embryo.
`
`The
`
`introduction
`
`is
`
`conducted with
`
`the aid of a microinjection system.
`
`When microinjecting the fused gene construct to an
`
`25
`
`embryo, an automatic microinj ection system which is
`
`able to automatically control amounts of DNA to the
`
`limit of 4 pl is preferably used because of it being
`
`18
`
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`

`WO 02/33063
`
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`
`superior
`
`in
`
`success
`
`rate
`
`to conventional manual
`
`microinjection
`
`systems.
`
`The mouse
`
`embryo which
`
`contains the IDPc fused gene construct was deposited
`
`with the Korean Collection for Type Culture of Korea
`
`5 Research
`
`Institute of Bioscience and Biotechnology
`
`(KRIBB) under the deposition No. KCTC 0874 BP, on Nov.
`
`4, 2000.
`
`3. Preparation of Transgenic Animal
`
`10
`
`Next,
`
`the
`
`embryo containing
`
`the
`
`fused gene
`
`construct is
`
`implanted
`
`into a
`
`surrogate
`
`mother
`
`to
`
`afford a transgenic animal.
`
`In the present invention
`
`the implantation of the embryo into a surrogate mother
`
`is conducted at
`
`the one-cell stage of
`
`the embryo
`
`15
`
`rather
`
`than
`
`the
`
`two-cell
`
`stage,
`
`for convenience.
`
`Immediately after the microinjection of the fused gene
`
`construct,
`
`the
`
`embryo of
`
`the one-cell
`
`stage
`
`is
`
`implanted to the oviduct of a surrogate mother, so as
`
`to reduce various processes necessary to culture the
`
`20
`
`embryo to the two-cell stage. For implantation into an
`
`oviduct at the two-cell stage, for instance, an embryo
`
`is required to be cultured for one additional day in
`
`an incubator.
`
`In order to implant a
`
`two-cell stage
`
`embryo
`
`to
`
`the oviduct
`
`funnel,
`
`the embryo must be
`
`25
`
`inserted deep into the oviduct, or it is necessary to
`
`perforate the oviduct by use of a needle. However, the
`
`implantation of
`
`the one-cell
`
`stage
`
`embryo
`
`to
`
`a
`
`19
`
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`

`

`WO 02/33063
`
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`
`surrogate mother may be conducted under conditions
`
`similar to those for qenera mouse embryos, al though
`
`the implantation site is the oviduct funnel.
`
`Using the transgenic animal thus made,
`
`the in
`
`5
`
`vivo activity of IDPc was examined in terms of the
`
`following indicators:
`
`1. Enlargement of Epididymal Fat Pad
`
`23 weeks after birth, F1 heterozygous
`
`10 mice had grown bigger than control mice. When being
`
`anatomized,
`
`F1
`
`heterozygous
`
`transgenic mice were
`
`measured to be significantly increased in the size of
`
`the epididymal fat pad with a body weight 14 times as
`
`heavy as that of the control mice. Additionally, when
`
`15
`
`being frayed,
`
`the
`
`t