Note: Descriptions are shown in the official language in which they were submitted.
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»ackgroumd o~~he Inve tion
1'tie invention relates to transgenic a~~s capable of socretiag
recombinant polypeptides in their urine.
'Fhe generation of recombinant proteins utilizing an animal as a
bioreactox hss the advantage of producing s recaa=bl~nant protein that is
likely .
properly folded. In addition, since anBnels can reproduce, they provide an
almost inexhaustible source of the recombinant protein. Ptoductton of '
recombinant proteins in animal fluids h2s been used widely, Examples
117 include the secretxvns of recombinant proteins in milk usitag milk
specific
promoters.
Urine presents an advantage over tha milk specific expression of .'
recombinant pdlypeptides for the following reasons: ~I) the process of
recombinant polypeptide production ~frotu urine is initistced irnmediataly
after
birth (or even prior to birth); (2) unlike the lactation process, urine-
specific
recombinant polypeptide production doesn't depend on a hormonal or
repror3uctivE status of the transgenic animal; and (3) both female and male
anvnals can be used fvr recombi~aant polypeptide production from urine, in ~
addition, trine naturally contains vexy small amounts of proteins es compared
to milk, thus facilitating the isolation of recombinant polypeptides from the
urine ef transgenic animals.
Tt has recently been reported that recombinant ptvteiris can he secreted
iu urine using the bladder specific promoter of the uroptakin II gene.
Speci-hcally, small amounts of recombinant human growth horrr~one (rc hGH)
Z5 were secreted in the urine of transgenic .mice (150 ~tghiter) under the
influence
of rhc uroplakir< II promoter (lCerr er al., Nature Bio:ec~tolo~;y 16: 75-79,
i
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secretion of
recombinant proteins using ale uropIakin II promoters ntay be hampered.
Y~ ti
The present invention provides a traz~gene that i~udes a desired
~lYpeptide whose expression! is driven by the uromodulin gene promoter, or
anothor proma~s from a gene whose product is speciiicaIly e~tpressed in the
kidney, thus allowing the expression arid secretion of the polypeptide from
the
kidney into the urine, from which it can be Isolated. ~ .
AceardingIp, lri a fit aspect, the invention provides tr~sgc~c non
human urine sarcting Futimals which are capable of producing recombinant
Polypeptides that are secreted extracellularly into the uci~ by the kidney
ti$sue
of the animal. The animals may be mamma, and may 6e rurninatyts, or non- .
ruminants, Representatives of non-hurctarl.u~e-secreting m~n~le,,~,seful in ,
~e ~n~erttion include, without limitati ,
~, rodents, rabbits, pigs. goats. sheep,
1~ horses, and cows, .
Tn one embodimetlt of tha first aspect of tk~e invention, t#~d recombinanE
PolYpeptide is an enzyme that is able to degrade or ca~yze a degrade,tion
reaccioo of undesirable components of urine (eg , ~~onra~).
rn a second aspect, ~e invention provides a method for obtaining urine
z0 of a transgenic animal t~ ~cludes the steps of: (a) generating a transgezuc
construct compost oP the controlling elements of a kidru"y specific gene
(including tho 5'~end promoter sequences and 3'-end elements) operably linked
to the nucleic acjd sequence of interest to be expressed; (b) screening the
constn~ct before a transgenic anima! is generated; this construct screening
z5 could be
Gone in kidney epithelial liras; (c) i~~ng ~~ ~e genome of a non-
human animal the transgenic construct; (d) collecting the eirine from this non-
hum~'m animal; and (e) isolating the product from ~e ~i~'s urine.
n~
.,~ '.~,, ~:xs. .~,~r~.~:
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Preferably, the kidney specific gene is the tu~oniodulin gene.
In a third aspect, the invention features a aar~sgene useful for the
generation of a transgenic animal, where the transgene trtckuies: (n) a
promoter
fI0Ir1 a kidney gpeeifC gene that is functional in the kidt~y sccretory cells
of
'~e aansge»ic animal of interest, (b) a leader sequence that is functional in
the
kidney secretory cchs of said tzansgenic species and (c) a nucleic acid
sequence encoding a roeorrtbinant endogenous or exogeno~ product.
Preferably, the kidney specific gene is a urvmodulin gene, Preferably, khe
tee' se9i~ce is operably linked to the nucleic acid sequence to form a
functional trar~ger~e rha,t is capable of directing the expression of the
secreted
recombinant polypepdde (encoded by the nucleic acid sequence) in kidney
secretory cells of the transgenic anirrrai, preferably, the kidney specific
gene
promoter is from the same species of animal as the trttansgenic animal (e.g,,
a
promoter from a goat uromodulin gene is used to generate a tc~.nsgeni~c goat),
In one embodiment of the third aspect of the invention, the Cransgene ' '
also contains is the 5' or 3' region at least one copy of an insulator
elvwncuxt
seduertce or a matrix attachmenf regiota. preferably, the trangge~ inci'udes
fog ~cdonal regions: (i) the insulator elernern sequence; (ii) a kidney
specific expression regulation portion (e.g,, the uromoduIin gene promoter);
z4 (iii) a lender sequence; and riv) a nucleic acid molecule encoding a
polypeptide
of znter°.,st~ The nucleic acid sequence encoding the polypeptide of
interest
rnay be cDNA or genomic DNA, or may encode more than one polypepnde, ar
a hybrid of two ditferer~f pc~teins cuith doruai.ns including two differenE
activiries (e,~r., hybrid poiypeptide contgis~ing the Fc porpion of an
imrnunoglobulin fused to insulist).
In a fourth aspect, the invention provides tuiz~ from a lransgcnie norr
human mammal, where the urine is choracterized by containing an endogenous
,:,M ~ .
a
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or exogenous ret;ombinant polypeptide, and is secreted by a transgenic
atlimal_
The transgenic animal is produced by introducing into its genome a transgene
containing a nutCleic ac,-id sequence encoding the roc:ombinant polypeptide of
interest, v~here the nucleic acid sequence is operably linked to controlling
S elements from a kidney apccific gene (e.g., the uromodulin get'le)- In one
embodiment, the controlling elemetlts are a genepromoter.
In a fifth aspect, the inve:~tion provides a nucleic acid molecule
inetuding ti) a nucleic orcid seqaxence encoding a polypeptide, fib a promoter
from a kidney specific getlo (e.g., the urorrlodulin gene), v~rhere the
promoter is
1d operably linked to the seauence, and ~ece the promoter is not naturally
associated with the nucleic; acid sequence, and (iiJ a leader sequenec that
enables secretion of the polypeptide by the urine-producing cells into urine
of
an aniirat. In various embodiments, the kidney-specific gezze is selected from
the group consisting of s caw, a human, and a rodent, and the animal is a
l.S mammal or is selected from the stoup consisting of a rodent and a ruminant
(e.g., a cow, sheep, or goat), The polygeptide may have biological activity or
may be soluble.
In a sixth aspect, the invec~tion provides an animal in which the gersome
of cells that convibute to urine production in the animal includes a nucleic
acid
20 molecule including (i) a nucleic acid seguence encoding a polypeptide, (i~
$
promoter from a kidney specific gene (e.g. , a uromodulin gene), where the
promoter is operably linked to the sequence, and where the promoter is not
natural)y associated with the nucleic acid sequence, and (iii) a feeder
sequence
that enables secretion of the polypvpade by the urine-prnduGing cells into
urine
25 of an animal. Ln various embodiments, the cells are kidney secretory cells,
tile
animal is a selected from the group consisting of a rodent and a r~sminarrt
(e.g..
a cow, sheep, or goat), and the animal is a mammal.
j'1.
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Irl a seventh aspect, the invQntiou featurrs a mead for producing a
~lYPeP~dd that is secreted in the urine of ate anir~.l, the method ineladir~g
the
steps of: (a) providing an embryonal cell transfeeted with a pvlypept~de-
~coding nveleic acid molecnlo operably linked to a kidney specific gene
promoter (e,g., a uromodulirt getle promoter) that expre~sea attd ~~~
secretion of the palypeptide from a kidney cell derived from the transfected
embryonal cell, where the promoter is not naturally associated with the
nucleic
acid sequence; (b) growtr~ the embryor,,al cell to produce an anirn~tl
Including
PolYPepbde expressing and secreting cells; and (c) isolatitt$ the protein from
the polypeptide expressing end secreting cel;s of the animal, preferably, the
animal is a mamrr~al.
In an eighth aspect, the inveneion features a method for producing a
poiypepdde, the method inciuding the steps of: (a) providing a c~h of axe
animal, the cell transfacted. ~swith a nucleic acid mvlecuie that cor~tair~s
(i) a
nucleic acid sequence encoding a polypeptide, (iii a kidney specific gene .
promoter (e:g., a uromodulitt gene promoter) that directs expression of the
i
polypeptide in the cell, where the promoter i~ not naturally associated with
tile
nucleic acid sequence, and (iii) a lender sequence that causes secretion of
the
polypeptide by the cell; (b) ovlturitag the iransfected cell; xnd (c)
isolating the
polypoptide from the cultmcvmedium of the cultured transfeeted cell. In
various embodiments, the cell is a kidney secrt'rtory cell or is an
irrunartalized
ceh, Preferably, the animal is a mammal,
In a ninth aspect, the invasion features a method for producing, in the
urine of a vertebrate, a recomhinartt proreiu that contains two yr more
subumits
Z5 ! inked >:o each other by disulfide bonds; the method includes the slaps
of: (a)
providing a transgenic vertebrate exhibiting urine-specific production of the
recombinant protein; (b) cailectang urine from the vertebrate; and (c)
isolating
',bla~ r ,7 r ~? s
Prit~lc,~
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~'e Protein from the urine. A related aspect of the im,~on is the vertebrate
a~'al used in this method. The ~thod can employ a uroplakin r, ucoplakin
Ill, or u~rornodulin pron~ter. In one preferred emhodi,rnent of the method,
the
protein, when it is excreted into the urine for tl~e ani,n~, is folded such
Lhat it
~ rendered biologically activ~ (f.e,, e,~~3ts at least some of the biological
activity pf the native form of the protein). Alternatfwely, in order to
protect the
~imal from possible deleterious effects oP the acti~re protein, the protein
can
be intentionally engineered to render it partially or wholly inactive at the
of secretion, but activatsbIe by simple means following aollectiort of the
urine
from the animal. Such methods are knoc~t and are described eg., In U.S.
Seria! N'o. 08/775,$42, commonly ~s:g~ti with the prc~,sent application.
Examples of rnultirneric proteins that can be produced according to this
ninth aspect of the invention Are monoclonal antibodies, of ar,y isotype; and
hetarodimerie fertility hormones. The train hormones in rhis category are
x5 follicle stimulating hormone (FS,~, l~e~~ng hormone (Z,~~ and human
chorioruc gonadotrophi~ (~CG). Each of tkiese ho~monos is, a glycoprvte3n
containing an alpha anti a bay subunit; the alpha subunit of all three is
identical, while the 6eta~,~,~~ differ and confer specificity of biological
action on each hormone. FSI~ and LFI $xe irnpa~nt ~~~i~ ~,~ue~ , .
~0 which have been purified from htunan urine_ These hormones currently are
made irt recombinant focal in cultured marntnalian cells. The sequence o f all
of these hormones are Iaiowrl. For example, p~ Application WO 90/02757
gives the sequences of LH anal FSIT.
The process of the
ninth aspect of the invention can also be used to
~ produce another ~:mpnrtant commercial hormone prodta;t, pregnant rxlare
serum
goaadotrophin (ppiSG~, which is a heterod3meric glycoprvtein containing ~
alpha and a beta ~u6unit. The me~od ;,an ~e be used to make inhabins and
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activins, which are also heterodimetic glycoproteiris that are produced in the
gonads. Mature inhibin consists of an xC-svbunit with either a ~A-or a ~iB-
subunit. Members of this family of dirner~s include iahibin A, inhibin B.
activin A, actfvin AB, and activln $ (which is a homodirner, a class of
multimeria proteins also included un the invention).
'The process of khe ninth aspect of the invention cea also be used to
make any of the multiple fortms of collagen, including homotrimeric and
heterotrirneric forms; the sequences of collagen chains ere known, and
disclosed, e,g., in PCT Application WO 96103051. The rnrthod ~Por the ninth
IO aspect of the invention can also be used to produce fibrinogen, wuhich is a
heterotrimeric protein whose sequences are known, eg., PCT Application WO
95rL2249. .
The invention also provides multiple transgenes encoding vazious
polypeptides or wersians of the same polypepHde (e.g., a polypeptide
1~ containing conservative amino acid substitutions, or amino acid
substitutions
that would enhance the stability of the polypeptide). These multiple
transgenea may ba coinjected (i.e., co-microinjecced) simultaneously, yr
sequentially. Thus, two or more recombinant polypeptides are secreted it the
transgEnie animal's urine.
20 By "prorein" or "poljrpepeide" is meant any chain of amino acids,
regardle.,s of length or post-tra~lational modification (e.g., glycosylarlon
yr
phosphorylationy .
Bar "naturally associated" is meant that two sequences (e.g , a promoter
and a polypeptide-encoding sequence) are operably lin(red in the naturally
25 occuzrlng genome of the organism from which the two sequences are derived.
For exgmple, the bovine uromodulin gene promoter is naturally associated
wid5 the bovine uramodulin-encoding seguenrx.
~~ ~ ~~t i ~. ~
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By '~not naturally associated" is meant tht~t two sequences te.g., a
grotrloter and a polyp~ptidarencoding sequence} arc not ogerabiy linked in the
ztaturaily occurring genome of trio organism from which one of both of the twa
88C~UeriGP5 61e derived. For e~cample, the goat uromodulin gene promoter is
nr,t
fi naturally associated with the bovine uromodulin-encoding sequence. In
adr3ition, the goat urornodulin geAe promoter is not naturally associated with
the human tPA~ncoding sequence.
By "lridney specific gene" is meant a gene whose groduCt is expressed
only in kidney calls. One preferred example of a kidney specific gene is the
uromodntin gene. Specifically excluded frarn the definition are the uroplakin
genes, since their produces are expressed in the bladder.
Hy an '~iz~sulator element .ecfuence" is meant a nucleic acid sequence
which, when operably linked to a regulatory element (e.g., a promotez)
directing the expression of a nucleic acid tnolecula of intarost on a
transgcne,
allows for the expression of tile nucleic acid molecule, regardless of the
position ef the genorrte ixf ~thictt the~transgone has integrated. 'I~gically,
an
insulator sequence is located immediately 5' to a promoter sequence.
By v "leader sequence" or a "5ianal sequence" is meant a nucleic acid
sequence which, when operably linked to a nucleic acid rnolecvle of interest,
allows for tho secretion of the product of the nucleic acid molecule. Z'tie
leader
sequence is preferably located 5' to the nucleic acid molecule. Preferably,
the ,
leader sequence is obtained either from s~.me gee as the promoter that is used
to direct the transcription of the nucleic acid molecule, ar is obtained from
the
gene from which the nucleic acid molecule of interest is deriYed.
2$ By a "transferred cclf° or a "transformed cal!" Is meant a call into
~rhich (vs inta an ancestor of which} has been introduced, by means of
reaombinanc molecular biology techniques, a nucleic acid molecule encodirtg a
~i~.
a
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poiypeptide of the i,nv~ention. Preferably, the cell is a eu'haryotic cell
from a
rrntlticellular animal (e.g,, a mammal).
lBy an ~~nbryoaal cell" is meant a cell that is capable of being a
progenitor to ah the somatic and germ~line cells of an organism, Exetnglary
embtyonal cells are err~bryonic stem cells fES cells) and fertilised oocytes.
.Preferably, the embryonal cells of the invention are mammalian etnbryonal
cells.
By '.germ line cell" is meant a eukaryotie cell. progenitor. os progeny
thereof, which is a product of a meindc cell division.
i0 By "operably linked" is meant that a nucleic acid se~u~ence and otae or
more regulatory sequeneea (e.g,, a promoter) are connected in such a way as tv
permit expression andlUr SeCretio~n of the product (i.e., a polypeptyde)
cnevded
by the nucleic acid sequanEx when the appropriate molErcules (e.g.,
transcriptianal activator proteias) are bound to the regulatory sequences.
By "endogenous," as used herein in reference to a gene or a
polypeptide, is rneartt a gene or polypeptiEie that is normally present iu an
~imal.
By "exogenous," as used herein in referP.nce to a gene or a polypeptide,
is meant a gene or polypeptidB that is not normally present in an animal.
?~~or
Z4 example, human growth hormone is exogenu~us to a transgenic goat.
By "tranagerse" is mint any piece of nucleic acid that is inserted by
artifice into a cell, ar an anrxstor thereof, and becomes part of the geaome
of
the animal wtaieh develops from that cell. Such a transgenr rrlay include a
gene w~hicla is partly or entirely exogenous (~;e., foreign to the tranagenic
ZS animal, or may represent a gene having identity m an endogenous gene of the
animal.
By "ttan~sgenic" is meant any cell 'crhich includes a micleic acid
CA 02343104 2001-03-16
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sequence that has been inserted by axti~'tce into a colt, or an ancestor
themof,
and becomes part of the genome of the animal which develops from that cell.
Preferably, the transgenic animals are transgenic mammals (e.g., rodents or
ruminants). Preferably thm nucleic acid (transgene) is inserted by artifice
into
the nuclear genome.
Hy "reporter gene" is meant any gave or nucleic acid molecule which
encodes a product whose expression is detectable. A reporter gene product
may twe one of the following attributes, without ruction: fluorescence
(e.g., greet[ fluoresc~nt protein), erazyrnatic activity (eg., luciferase or
chtorampheruicdl acetyl transferase), toxicity (eg., ricin), an ability to
confer
resistance to a reagent (e.g., resistance to neUtnycit, by the neo gene or
resistance to copper by the metalloth3or~ein-eticodirxg gene), an obility to
confer
susceptibility to a reagent (e.g., 9asaeptibility to gancyclovir by the
I~ecpes
Simplex Virus thymidine lZinase-encoding gene), or an ability to be
specifically bound by a second molecule, suc<1 as biotin or a delectably
labelled antx'body te.g., binding by biotin by the avidin-encoding gene or
binding by an antibody (gig, , detestably labelled) by a cell surface
expressed
epitope-encod;ng $ene),
Eria'f Description of h l~rawir~,gs
~0 Fig. l shows the partial DNA sequence of the human urornodulin gene
promoter (SEQ >17 NO:1; GenBanlc Accession No, 575968; Yu et al., Gene
Fxpr. 4: 63-?5, 1990.
Fig, 2 shows the partial DIVA secjuenee of the bovine uromodulin gene
promoter (SEQ xD N0:2; GenBank Accession No. 575951; Yu etaL, suprr,~).
Fig. 3 shows the partial DNA Sequence of the tat uromodulin gene
promoter (SEQ ID N0:3; Gen$ank Accession No. S~59d5; Yu er al., sr~wrcx).
,,
I
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leg. 4 is a schematic diagram illustrating an example of a method to
generate a urornodillin promoter ttansgenic construct. The nucleic acrd
sequence (flaziked by BamHI and SpeI sites in this fzgure may encode human
tPA (see Fig. 5), or may encode a reporDcr gene, such as lucifetase.
S Fig. 5 is a sct~~tnade diagram of a PCI~ reaction to generate a human
tPA-encoding cDNA fragment flanked by ti Ban>l~ recognition sequence on
the 5' end and a Spel recognition sequence on the 3' end.
> ig. 6 is a achem&tic diagr~un of a tr~nsgenic construct that includes a
goat zxromodulin gene promoter dircctiag the expression ~of a human tPA-
~0 encoding sequGxice. The backbone plasmid has a hygrarnycirt resistance gene
foz eukaryotic cell selection, an ampicillin resistance gene fc~r
prok2~.ryotic cell
selection, and a ColEl origin of replication for ampliflcatian in bacteria.
I:igs. 7-12 ate schetnatlc represenrations of the cotsstruction of
expression vectors of the invention.
15 Fig, x3 is the sequrnce of the goat Ub~ promoter f ~E(,7 B? Nt7:4).
i?etailed Description
'Fhe present invention relates to a process for racereting recombinant
proteins in the urine of non~human anirtsa~Is. This process uses expression
20 vectors containing promaCer'sequences based on the regulatory elements of
uromoduIins (also called the Tarttm-FTorgfall glyoprotein ('TITS) f
e~romucoid),
or the promoter sequ.,Rncess fxom other kis~ney-specific genes to express
recombinazit proteins in the kidney, thus allowing thei: seGreuon into wine.
Uromodulin is synthesized by the kidney and Iocalixes in the early y
25 distal tu6uia and the renal ascending limb. ~rornoslulin is the mast
abundant
protein in human (30-45 mgl~ hours) end rat urine ((1.5-z.9 mg/24 bows)
I~okhale et al., Ilro~ Res. Z5; ~4?-35~4, 1997y. No uromodulin protein has
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been detected in normal tissues other than the kidney bowie, A.J., J: pathol.
153: 399-404, 1987), however truss-reacting proteins with antibodic5 against
uromodulin have heen identified in l~nan and rat sera at Iow-levels (Lynn and
Marshall, Biochem. J.194: 561-568,1981; Wirdnarn and Milner, ~Vephrori 4a:
362-36?,1985), Antibodies raised against uromodulin crossreaet arith the loop
of ~enle in the kidney of mammals, superficial layers oP the skin of several
amphibians and fish, superficial layers of the oral mucoea and gills of fish,
and ,
the distal tubules of the kidney of amphibians, No erase reaction is observed
in avian and reptile species (Howie er al., Celt Tissa~e l2es Z?4:
ISrl9,1993).
Uromodulin i9 a 6lb.amtno acid, 85 kD~. gIycoprotein with fn vitrn
irnrnuno-suppressive properties. The partial bovine anrl rodent u~modulin
promoters have been cloned and shown to contain the typical et~ntrolling
transcriptional elements in the proximal promoter (Yu et al., Gene Expr. 4: 63-
75, 1994). Using tha uromodulin promoter is useful for generating urine-.
secreted proteins because, since the level ofi,nuomvdulin can be increased by
increasing the urine volume, a means is thus provided for itlcraasing the
total
output of the recombinant product, whose expression is directed by the
uromodulin gene promoter. In addition, 5inco uroniodulin is secreted in the
urine of the fetus, amniotic fluid sampling may allow early detection of a
ZQ trausgenic fetus expressing'a recombinant (rc) Polypeptide whose expre~ien
is
directed by the ummodulin gene promoter (see the procedtue of Phimister and
Marshall, Clip. Chim. Acts I28: 261269,1983).
a .c ..
For the ge~exation of a transgenic construct that allows for the sacretioa
2S of a recomhinant polypeptide from kidney secretory cells, any appropriate
backboae may be used. '~l'here the nucleic acid sequence encoding the
.. " ~ , ~ .; ,
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recombinant palypeptads of interest is genomic DNA, the backbone p(.asmid
may be derived from a cosmid (e.g., SuperCos or pWEI5. both commercially
available frpm Stratagene, ~,.a JoDa, CA). ,
Preferably, the backbone has a prokaryotic origin of replication, as well
as a gene encoding a selectable marker that may be used for prokaryotic cells
(e.g., 2znpicillin, tetracycline, and chloramphenicoD, for easy propagation
and
amplification in transformed bacteria. Prior to lnicrofnjectian, the complete
trsnsgenic construct may be linearized by removing all the bacterial
sequettceg
(i.e., the bacterial origin of replication and the hacterlal selectable marker
genet .
In addition, the backbone plasmid should have a selectable tnadrer gene
that may bo used for solcction in a eukaryotic cell (for example, hygromycin,
neomycin, puromycin, and neomycin). Such a marker gene may be unaar the
expres9ion of its endogenous promoter (e.g., the puromycin-resistance geane
1~ promoter directing the expression of the pvromycin-resistanc~ gene).
A.Iternetive3y, a relatively weak promoter (e.g., the Sy~l~ early promotes)
may
be use to direct the expression of a selectable marker gene product.
Representative polypeptides encoded by nucleic acid setluences to be
expressed and secreted in the urine iztclude, without limitation,
erythropoietin
(EPO), human tissue plasminogen activator QatPA), insulin, antibodies (e.g.,
monoclonal or humanized), and hormones (e:g., human growth hormone).
The basic trariegeuic construct contemplated includes the following
S
elements: the plasmid backbone; a kidney specific gene promoter (e.g., the
uromod'islin gene promoter) operably linked to a leader sequence and a
nucleic acid seduence encoding a polypeptida of interest; and a
polyadenylasion signal located 3' to the stop codon of the nucieie acid
sequence. Tlle lender sequence may be derived either froze the gene whose
fl~ .' ~ . ~ '
." ~raW a x,d,. ~.
CA 02343104 2001-03-16
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prortiotec is being employed, fmm the nucleic acid sequence, Qr from alt
alternate secreted protein-encoding sequence (e.g., ~ I~ genej. Likawisa,
the 3' UTR, a~tdch includes the polyadenylation signal, rnay be from the gene
from which the protndter is derived, from the nucleic acid sequence of
interest, or from a~~t alternate soiu'cx Ce.~ , the SV40 vitas). A 5' UTI~ may
be
located bet~areen the promoter sequence and the leador sequence, tend may be
from the gene whose promoter is being employed, the nucleic acid sequence
of interest, or from an alternate sourco. For example, 'where no convenient
restriction enz3me recognition sequence exists in a nucleic acid seq~,ience of
inte.~est between th8 5' -CT'~',R, the leader sequence, the nucleic: acid
sequence
(l.e., the coding sequence), and rbe 3' UTR (whiW includes the
pol~radenyJation signal), the tr$nsgenic conatnu;t may be generated an a throe
part ligation of the linearized backbone plasmid, the kidney speck gene
promoter seduence (flanlted by appropriate linkers), and the following .
fragment, likewise flanked by appropriate linkers: 5' IfT'R, leader sequence,
nucleic arid sequence encoding the poIypeptide of interest, and 3' U'J'R.
The a ' Crane o er an E
~omoter
The partial sequences of human, bovine and cat uromodulln gene
promoter have been described (Figs. 1-3, Yu et al., s~upn~), Tn addition, the
GenBa~ak sequence database provides a number of uromodulin sequences from ~
~ a variety of mammals, including human (Accession Nos. M1S881 and
Ivi17778), Us~g ~~~1 molecular biology techniques, the seqc~a~ce of the
uromodulin promoter from a pa~c~ar a~~l (e.~ , a 80~) ~y ~ isolated ,
from genamic L~NA from chax animal using standard library screening . .
te~chni~q~ues see techniques in, e.g., AusubeI etal., supra).
CA 02343104 2001-03-16
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In addition, since only Bart of the promoter sequence has been
elucidated. the remainder of tlae promoter may be derived using standard
primer extension protocols or a PCR-based "gene walking" techniquE (~usir~g,
for example, the (ienomeWalker'TU trits c.ommercialiy available krorn
Clontect~
Laboratories, lnc., Palo Alto, CA).
Once the uromodulirl gene promoter is identifiexl, it can be aperably
linl~ed to a reporter sequence, such as a sequence ertcodirg lucifer~e, This
reparber construct may be cued to test the ability of the cloned pxorcloter to
express acid secrete lucifexaae from t~ar~sfornled cells. For example,
following
~ransfomlation of kidney Cells (e_~ , COS tails) with a coastrttct of the
umrnodulin gene promoter opcr8bly linked to the luciferase encoding
soquence, the c~.lture rnedifl of the cells may be quickly a8saped fomhe
preseztce of luciferase (using, for exarnple, the Iucift~se detection assay
kit
commercially available fron~a Promega Corp., lViadison, WI).
~rpey~tide ~1 ifL L~~~on
Onrx the recombinant pro#ein is expressed ,in the urine, it can be
purified using standard protein purification techniques, such as affuuty
cluomatography fee, a.g., Austrbei et al., ~~~nt p~to~ol5 in Nioleeula'r
iol Joha WilGy & Saris, New York, N'Y, 1994). In an e:cample where the
recombinant protein is human epidermal growth factor (EGG, the urine may
bC added to arl affinity column to which are immobilized azita-human EGF
antibodies (corrunercic~lly available from, for eagmple, LTpstete Biotech.
Inc.,
Lake Placid, i~'Y). Once isolated, the recon~birant protein can, if desired,
be
further purified by e.g., high performance liquid ehrorrtatngraphy ~FpLC;
e.g.,
sea Fisher, La r or,~ Techni4ues Ir1$io_Chem'~.~~~,r~jnd ~olee~,lpr ~ o V,
ads. Work and Burdon, Elsevier, Z98~J).
;, ; . ;
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-16-
C',Zet~e atinn of a rans~nic Anj~i
Trarrsge»ic constzucts are usually introduced into cells by
microinjection (Ogata et ~L, U.S,P_N_ d,8i3,292)_ A microinjectad zygote is
then transferred to an appropriate female resulting in the birds of a
transgenic
or chimeric animal, depending upon the stage ef developme'nt of the zygote
when the transgene integrated. Chimeric enirr~als can be bred to form true
gerrr~Iinc transgcnic animals.
In same methods of trangenesie, trsmsgenes are introduced into the
pronuclei of fertilized oocytes. ror some animals, such as mitt, fertilization
is
performed in vivo and feztilized ova are surgically removed. In other animals,
tt~e uvu citn be removed from live, or from newly-dead fe.g., slaughtrshousc}
animals and fertilized in vitro.
Alternativelir, transgenes can be introduced into embryonic stem cells
(FrS cells). Transgenes can be introduced into such cells by electroporaEions,
microic~jection, or any other techniques used for the tt'artsfection of cells
which
ere known to the skilled artisan, Trar~sformed cells sre combined with
blastocysts f cam the animal from which tllep originate. The cells colonize
the
embryo, and in some embryos these cells form the germline of the resulting
chimeric animal (~aenisch;~R., Science 2da: 1468-'7.474, 1988).
A.itefnatively,
ES cells can ~ used as a sowce of nuclei for transglantstion Into an
enucle3tea
fertilized oocyte, thus giving rise to a tr~genia aaimal.-
ultlple Trar~~,genes
In accordx~x~ce with the production of recornblnant protein in the urine of
a tranggeric animal, where the protein is composed of two different sub~units
non- covalently bonded to one another, it may be desirable to produce both
subunits from the same tratiegerlic animal. xn surh a situation, taro
different
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transgenic constructs, each encoding one of the two subunits, may be
sirnultaneausly or sequentially co-rnicroinjected into the same xygete to
produce a transgenic animal ezpressirg both wbunits.
Alternatively, nucleic acid sequences encoding each of the two subunits
can be cloned into the same expression cassette wlth the insertion of m
intervening n'bosom~ entry site (TRFS) (fang etal,,J: Yirol.62: 2ti36-Zd~3,
1988; gurtu et al., Biockem Biophvs. ~:es. Camm. 229: 295-X98,1996). The
advantage to cloning both nucl~ic acid sequences into the same exgr~ession
cassette is that only one single construct is needed to, generate the
transgenic
animal.
It will be uruierstood that the pro~:edutes described above may be also
used to generate a transgenic: anirttal secreting twv full length, ucirelated
proreins (e.g., a recombinant insulin potypeptide and a recombinant human
tissue plasminogen actlvator polypepcide).
Pre-I ' 'on Sc
Prior to the microinjeetian of a trar~sgenie cflnstruet encoding a desired
poZypeptide, the construct may be screened in cultured ltidz~ey cells i~ v~rra
fox
an ability to encode a polypeptide that is expressed and secreted by the
transfected cultured cells. ~ultwed kidney epiibelaal Delis, such as COS cells
oc MDCK c~lIs (both comrnerciall5y available frotri the American Type Culture
Collection (ATCC, Rockville, Mp)), may be transformed with the transgenic
construct usizig any standard trartsforrnatlpn protocol (e.g., CaP04
precipitation, DEA,F~dez~aa, electrorporatian~; see Ausulxl etal., s~prta).
Since the kidney specific gezte promoter (eg., the uromoduliu promoter) is
active in these cells, the desired polypeptide encoded by the transgenic
construct wdl be expressed and secreted by the transformed cells if the
a
~'~.'_,~''~k .. Y~.
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-18-
tras~sgerzic construct is functional, The conditioned culture media of the
transformed cells may thezi be asswyed for the presence of the secreted
recombirsant polypeptldo.
Itc~tero S,c_teenin~g,
Smaller mammals (e.g., rodents) have ~t reasonably short gestation
s
period, thus allowing a rapid detorminatian of whether or net the trar~genic
animal Is truly ixansgenle and able to prodeice the recombinant protein in its
urine. I-Iowewer, for larger animals te.g. , cattle), it may be desirable to
determine whetlZer or not the fetus is indeed transgenic and capable of
pmduci~tg the recombinant protein in its urine prior to birth.
Fetal renal function starts Barry during gestataan and uromadttlin is ,
detectable in the amniotic fluid, implying, of course, that the urvmodnlin
promoter is active, fierce, since ttte feta~s secretes urine into the amniotic
fluid
of i~ placenta, sunniotic fluid rrtay be removed and tested fot the pzesence
of
the zecotruirlant polygeptide whose expression is directed by a urornoduiin
promoter, Such testing may be by any standard immrrlunolvgical assay te,g.,
ELTSA. Western blotting anal,ysi9), or, if no specific antibodies are
available.
by purifica~.on of tire recombinant polypeptide and N-terminal sequencing.
The following examples are to illtzstzate the invention, and are not
meant to limit the inveation i,ta any way. In addition, although the following
examples describe the urnmodulin gene promoter from the goat, it will 6e
understood that a uromodulin gene promoter from another species is also
contemplated by the invention. Furthermore, other kidney gpeclfic gene
promoters, whether thiey direct the expression of art intracellular or
secreted
protein, are also within the ittveation. 'Where a kidney specific gene
promoter
that directs the expression of a nvn-secreted protein is used, the leader
i
~S i.
.;s
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-19-
sequence may be from the urornodultn gene, the nucleic acid sequence
encoding the desired polypeptide, or any other secreted polypeptide.
The uromodulln promoter sequence ~y be cloned usi~ s~andaid
techniques (gig., bybridi2ation under non-stringent conditions) to isolaoe a
urorpodulin promoter sequence ,ping ~ a p,~be one of the lodo~rn partial
urornodulin promoter sequences (~ e. , the rat, human, or. bavfne sec;uenae~,
pf
course, the animal desired to be made transget~ .will affect which of the
i
knowtz partial sequences Will be used a5 a probe, For example, should a . .
1(? transgenic goat be desired, the Dartial sequence of the hovine uromodulin
F~~'na~r (provided in Fig. 2; Yu et al., supra) may be radiolabelled and used
to probe genomic Di~TA prepared from goat tissues generated according to
standard techniques ft-orn goat cells). Should goat uromodulin prorrioter thus
isolated t~ flat to be less toast the full length promoter, the ~1 length
:t 5 promoter may be isolated by ext~erldittg the isolated fragment using
primer
extension, :~ltematively, tho full length promotec msy be obtained using ~
Genome W~kerTM ~~ commercially available from Clontech.
Once the goat uromodu~ia promoter se,~uencv has been cloned,
commercially available linkers (commerci$(ly available, for example, from
~0 New England Hiolabs, Beverly, MA) may be attached to the ends of the
promoter sequence at~d ligated i~ a bacterial plasmid containing a bactcriel .
.
ongtn of :eplicatian (e.g., the pl;TCl9 vector) for rapid amplification of the
promoter in vector~transforrned F toll. Thus orriplified, the promotes may be
freed from the pU'C19 vector by dagestiol~ with the restriction endanuclease
MS w~ch specifically cleaves at the linker sequence, and be subeloned into the
transgerzic cortstrttct.
' h
CA 02343104 2001-03-16
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Alternatc~ely, the digested fragment can be combined with digesfied
fra8~nts corresponding ro the leader sequence and the nucleic acid encoding
the desired polypeptide (e,g., tpp~~ and used in a fo~part lig$tion with a
EcoRIISpeI linearized euhatyotic expra~sion vector, a f~ragrneat con~ta.ining
the nucleic. acid sequence of iu~tcre$t, and a fraga~eret contaiaLig the 5' I~
and leader seque~tce (See Fig, ~), zt y4r~1 ~c ,~~.s~od Sgt in the scentario
of
Fig. 4, a four part ligation need be used only ip ~e ~~ ~ and leader
r
sayuence are not from 'tl~e tirarnodulin gene ar the human tPA gene. Note that
the backbone plasm:d in FYg. 4 already contains a 3' UTR attd
polyadeny7aiian sig~13' to ifte stop colon of the insexted human tPA-
encodir~$ nucleic acid sequence.
If a transgenic goat is desired to produce human tissue plasminoger,
tS activator (tp.A), the coding sequence of the tPA gene may be generated ~~g
rye lo~own human tPA CDNA sequence (Qe~t~ark Accession No, E02027),
As dQpic~,d ~ Fig. 5, a forward primer containing a Bad restricxion
enzyme recognition, site at its S' end and a reverse primer containing R SpeI
restriction enzyme ret;ognition site at its S' er~d may be used to PCR amplify
a
human tpA_$ cDN,4 Sequer~e from a human cDNA library ,
(comrnetctally available from, for e,~tample, Clontech, Palo Alto, t.".A). '
Fnliowitag amplification, the PCR product may be dige$ted with $snr~ and
SpcI and, as above, ligated with the fragments corrPSpotrding ~ ~e
'~rvmodulin prompter and the leader segu8nce with the F.coRI and Spec
lineacized vector (see Fig. 4).
.,.lie '. ~ ~~' ~S L ~u ~ .1~~ :A
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-21-
I~ior to injeet~g gflat zygotes with tile tz~u,s$~c cons
tract shown on
Fig. 4, the ability of the cor~truct to ez~tble a~tra~fo~e
d cell to exFres5 ~
secrete a polypephae ~codod by the eor~sttuction ma
Y first be deterrr~ned
using transformed cultured kidney eprth~~ cells. 1n this
example, greep
mo~eY kidney ce118 (CO$ cells) are transformed with
the ~~
~~~ transYormation mptt,."a T~ ~ _ _ . . ~t usin thd.
-..~ ~~~ wnswuct may fist be
~~xed trsxng $ urriquo restriction endonuclease taco
~~on site located
within the bacterial origin of replication or the p~k~,o~c set
gene. . ectabIe maker
0 Twenty-fovrhours following uansfQm~ati4 . ,
n, the cells spaat
conditicmed media fs exchaogEd with fresh media, and the rolls
to cn(mre. The
~e i"etumed
next day (~,e. , dg hours follo~~ing transfextio~, the
~~itioned media of the cells is re,~~ed arx! 2asayed f~ the rese
F nCe of
human tP~, using D~estern blotting analysis with a human tpA_
specific
I5 an~'~dy ~ a p~be. If desirable a
9 titles of tPA are pro~ced by tree
tra~forrned ce~Is, the construct rnay be used, as is, to microin'ect oa
J g t
~3'gates. Icy the alter~dve, where tPA is produced by tile transformed c
alts,
but in a qu~tity tf~at is less than dean-a,blc, the construct ~ ~ .
Y modified,
and rete~yted in COS cells.
i
After repeated screening in Cpl calls, a n.ansg~c ~~~et
sueH ~
hex sbowa schematically irt l?ig. b, may 6e generated. The cone
tract spawn
has ~ ~s~ator eleasez~t seqii~ce located up~t~, of ~e oat
8 uromodu~lin
Fromflter sequence to $lIow exprnsax~ of the construct reg~~~ of the sit
i
of integration. Itt addition, the Sy~p 3t QTR which ~ca
ude~ the SV40 pv,lyA,
signal) of Fig. 4 is replaced arith the 3~ ~T~g of the goat uroniodu ' . .
hn gene.
CA 02343104 2001-03-16
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The construct may now be linearized, if desired, by digesting the consuu~ct
with xhoI and X6a1 to remove the ampicallitx resistance gene and ~s ColEl
origin of replication (f,~, the digestion fragment that includes the ColEl
origin and the ampieil lin resistance gene is disc;arded). The ~main;ng
fragment (i.e,, ~e ~nsgeue) is next used to microinject goat zygotes,
it will be understood that in the schematic diagrams of Wigs. 4-6, the
Particular restriction endonucleases are exemplary; any suitable restriction
endanuclease may be employed. Ju particular, where a restrict;an
endonuclease is desired to be unique (e.g., to facilitate cloning end
subcloning
of ~e goat urornodulin promoter), the linkers used Co flank the soqt~er~ce may
be recog~tion sites of taro-cutting e~y~s (gig., SseI or Nato.
The construct depicted in rig. 6 cars be used to generate transgertic
mice capable of secreting human tPA into their elrine. The generation of
Qansgenic ;nice priUr to the generation oP Iransgenic goat' is preferable not
Only because of the g~teater time expenditure (r: e. , longer gestation
period)
r~~'~ to generate a trarzsgenic goat as ppppged to a transgenic mouse, hut
also because of the higher expense in maintaining and housing the animals.
Hence, mature female mice 'are st~perovulated and mated with tziales to
produce fertilised eggs. The eggs are harvesr~ for prnnucleat
microinjection, For example, a Laitx micro-manipulator and a Nikdn inverted
micm.scope may be employed far the mictoinjecti~ns. Pseudopregnant
female mico are t6en'implanted with microinjected two-cell err~bryo8, Once
~e pays are barn, their t~rme is screened for the prese~e of human tPA.
~',~~~ple
_ i .
4
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Expre55ion of the monoclonal antibody K20 can be achieved under the
control of the rnot~e uroplakin II gene pzomoter is a eukaryotic expression
vector, pcDNA4lHisMax.
K20 is a rnause monoclonal nntihody ("mAb") that recognizes a
particular epitope on human CD29. A soluble form of K20 was shoovrt to
bloclr peripheral T eel! gctivation and proliferation induced by an anCi-Ci)3
gntibody. This negative effect might by mediated by an increase in cAMP
levels or an inhibition of dlacylglyce:ol and PA formation. The tn vitrn
ftinet3anal effects of K20 make it a good candidate far therapeutic
1 Q imrrmnosuppression, A humanized KZO mAb (i~u-K20) has been produced
with potentially reduced immunogenicity nod functional, properties identical
' with the marine nulb K20 (Paul, M'A. et al., Mol. Im»=unol. 32:101-116,
1995).
A group of membrane proteins known as uroptakins, produced on the
apical surface of the bladder urotheiitun. can; fot'rn thick protein particles
making two-dimensional crystals (t>te "urothelial plaques") that cover over
ii0% of the apical surface of urothelium (Yu, J. et al., J. Cell eloX 125: 171-
iBZ,199d; Sun T. T. etal., Mol. Bio. Rep. 23: 3-11,1996). They are
urothelium-specific rna~cers and are conserved during mammalian ovolution
?0 (Wu, x. R. et al.,J. Blob Chern 269: 13716-13724, 199d). Recently, using
the uroplalEin TT gene promoter (U.S. Pat. No. 5,8?~,543), transgenic mice
thot
express hicman gro~tvth hormone (hCxH? xn their bladder epitheliwn were
ge~rated, resulting tn the secretion of the recombinant hGH into the urine at
100-Sad nigh (Kerr, D.E, etal.,Na~ Biotechnol, 16: 75-79,1998).
Described below is ~e construction Qf the Hu K20 expression
cassettes tiring a eukaryvtic expression vector, pcDNA~IE~sMax (Invitrogen).
Standard methods are used fox plesmid purificasion, rrstricrion enzyme
I
CA 02343104 2001-03-16
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1 ~ ~ '.
digestion, DNA ligation, and DNA ~zagmeat isolation.
Feferring to Fig. ?, constsuciion of the Hu-K20 light chain expression
cassette, PCR is performed t~it~.g DNA of the 4-83 wkmMi6 expression
vector (I7t'. Zhou, lVexia) as the template with a S' seQSe primer
(5'GCGCAGGCC~CCGCTCTAQAC~CG3'; SEQ iD No:5)
contfcining a Munl site (tmderlined) and a 3' anti~ense prlrner
(5'GCiGCAGCTCt~AGtfTCC~ACGCCCCATCCTCAC3'; SEQ XD NO:~
containing a Xhol site (underliner>3. Thn 2.4 kb amplified fragment from
upstream of the chicken p-globin gene, used as. an izlsulator, is digested
with
Mvr~1 and XhoI, arts! ligated~at the Xhar site pith a 3.6 kb-long~hol-l~em~II
fragment of the UPII gene promoter, released from pGI containiag ihc
genonuc sequence of the 'UPII (Dr. T. T.Sun, Kaplan Comprehea~siE a Cancer
Center, New York University School of lVladiclne, New Yor3~. A
pclaNA4~'HisNfax expression vector (lnvitrogen) is digested with Mur~T and
Barn.HT, and the CMV promoter-les9 vector is ligated with the Munl-BanaHT
combined fragment of ttse insulator and the UPn &ene promoter. PCR is
pcr'ft~rmed using the excised lvlt~-BanaHI fragment contain'it~g the SPlb3
sequencos ~(Garman, C.M. et aL: Proc. NatL Acaat Sci 79: 677 i-6781, 1982)
from pcDNA~lI3isMax as the template with a 5' sense prIttner
z0 (5'GCGTAT~~TCCAC~G~Ca,GAGGCTTG3'; SEQ 1D N0:7) containing a
EamIdI site and 8 3' antisense primer
(5'GCGT.ATTGCA _,T'~GC~'TTCGGAGGCCGTCCG3'; SEA IJ~ 1~I0;8)
containing a $swiI site. 'I'he amplified SP 163 fragment, which servos as a
trzanslatianal eahuuc:et (Gorman. C. M, er ail., Proc. NatL Acad ScL 79:
6777..
6781, ~98Z), is digested with Bam~-1I and Bswi. A 320 by fragment of tk~e
v ariable region vfi the mAb Hu-K20 light chain mod~etle is obtained 6y PCIt
amplification of pSVhyg-I-lu'VhG~O-HuCK plssmid DNA (Paul, M.A. et aL ,
.,
. . , .a ~ t
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.
-25-
Mod. Immunol. 32: 1~1-llb, 1995) with a 5' sense primer
(5'GCGTA'1'_I'GCAT"Ta~C~ICCATGGGATGGAGCTG'I'ATCATC3'; SEQ ID
N0:9) containing a Bswl site (uaderlinedy, the Kozak sequence (italics) and
the start colon (bold) followed by a partial mouse V4'l Ig heavy chain signal
sequence, arid a 3' antiserzse primer
45'f"rCGTATC~~,~ACTTACGTTTGA'FC~'CCAG3'; SEQ II) 10:10)
containing a HpaI site (underlined). T'bte amplified product is digested with
Bswil arid Hpal. Another PCR is performed in pSVh~g-HuVKK20..1'iuCK
with a 5'sense primer
I4 (5'CGCTATG~,CGAC~'AGAC'I~t'AAACACCATCC1"QTI~'CG3'; SEt.~
1~ k~10:11) containing a HpaI site (underlined a splice donor signal (bold)
fnr
the 3' ertet c~f the variable region of the light chain, and parrlal 5'
sequence of
the lZUman genc~mie Ig kappa cotistaixt region, and a 3' antisense primer
(5'GCCTATGT'I'TAAACGAGTA,GT'fG(~TAAACAACAG3'; SEQ a1 NO:
12) containing a pmeI site (underlined) a»d partial seQ~uec~e of tfle hwnan
genomic Ig kappa constant region, The PCR product is digested with HpaX
arid Pmel. The amplified PC~t products are ligated together through Bswil
and HpaI sites, respectively. The peDNA4/I-I'~slv(ax/rnsulator/UPII is
digested
with Baml~ and Fmel and Li.gated with the ligatcd PCk product with
owerhartgs of BarnH'I site a~ 5' arid prnep site at 3' to form
pc3?NA~INC~fJPIj-
IC20L (Fig. 7).
The expression cassette pcDNA4lMax/~TPIT-ZC2flL can be used as a
versatile system fur the r~oning and expression of irnrnunoglobuli~
cor~isring of heavy and light chains. An advantage of this system is that both
the V-genes and the C.genes may be exchanged as cassettes in the vectors, '
given the low frequency restriction en2ytne 53tes in the Ig genes that have
bean chosen. Xn addition, The V-genes can be lrept intact, and transient and
CA 02343104 2001-03-16
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-26-
staple expression of antibodies cazz be done eithec from two soFaratc rectors,
or trorn one tandem vector (Norderhaug, L. et al., ,!. Imm;unaL Metlsvds~
2(14:
77.8?, i99?).
Referring tp Fig. 8, for tl~e constractipn of the Hu-K20 heavy choirs
expression cassette, a 360 by fragment of the vaZ'iab~e region of the Hu-K2fl
heavy chain module is obtained by PCR amplification of pSVgpi-YHK20-
I;IuCyI DNA EPaul, ~,p,,, er ai:, Mdl. Immunol. 32:101-lld,1995) with a ~'
sense primer ,
(5'GCGTA GC CCACC~CGGATOCiACCTGTATCA~'C3'; SEQ lb
ld N0: 13) contairung a I3swil site (underlined), the Ko4rdc sequ~acx (italic)
arid
the start eodon (bold) foho~cved by a partial mouse'V47 Ig heavy chain 9igrtal
~9~e. end a 3' antisense primer
(5'GCgTAT~TC~TCTG,A,GGAGACGGTGACCGTG3'; SEQ zD NO:1d)
conraining a Clan site (underlined). The annplified product is digested with
Hswil end CIaI. Another PCR is performed in pSVgpt..VI3KZb.~IuCyI with a
5' sense primer
(5'CGCTATATCGATAGGTGAGTAC~CTTTC'I'G(~pGCAG3'; SEA TD Nn;
15) tong a Clan site (underlinedy, a splice donor signal (bold) for the 3'
end of the variable region of the heavy chairs, and partial 5' secfuence of
the
hurnarz genotnic Ig garnrna-I ~comstaat region, and a 3' antisense primer
(5'GCCrTAT~~ACGACCC(3C'TCTCrCC'TCCCTC3'; SIrQ ID Na: r6)
containing a PmeI site (underlined) and partial 3' sequence of the human
genamic Ig gamzna..l constant region. The PCR product is digested with CIaI
and PmeI. The two unplified i'CR products are joined through the CIaI site. v
The pcDNA4lMax/fTpII K20L vector is digested with BswiI gnd PmeT atld
ligated with the ligated PCR product with overhangs of Bswil sate and 5' and
Pmel, site at 3' to form pcDNA4/IVIa~cliJpn_K20H (Fig. 8).
CA 02343104, 2001-03-16
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K~.~ w' F JF ~~,;~ it,1? ; ~ ~ ~b EFh IdUEPIC3EN +~9 89 ?399~~65 ~;b.. . ? ?~
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_Z~.
A co-expz'ession vector containing bath Iight chain and heavy ci~ain
modules is generated as follows (Fig. 9). The pc;DiYA4lMaxlUFII-KZOH is
digested with Xhol and Ptnel, 'Z'he digested XhoI-Pmel fragment is gel-
pnrifieci, the X~,ol sticky end is filled with Klenow, and blunt-end ligated
to
the pc,UI~tA4IMaxIUPII-K20L alroe~dy digested'witt~ Prilel, to form
pcDNA4lMaxlflPn-K20LH. The orientation of the pcDNA4IMaxIU1?II-
IC20LI is verified by the digestion of the vector with Hpal and Clal.
A human urothelium cell line (hu609) (Stacty, S.D. etad., .l~Io~
Carclnog 3. 216-225,1990) is to be used to assess thG established cxprass3on
cassettes. The DNA of the L chain anti the H c&ain constniets is purified by a
Maxi-preparation (SOP#008) and as introduced simirlraneously at equal molar
concentration iota the Hu609 colt Jinc by standard transfection techniques.
Culture supertlarant is precipitated and appli ed to an anti-human IgG (H
chain
specific)-agarose column (5igrna). Protein cancentratian of efut~d fractions
is '
assayed by Bradford microassay (Hio-Itac~ and frectlons containing proteins
:ue checked on a 10°Yo SDS-PAGE. The expressed proteins are also
detectEd
by Western bloc with anti-IgG CH chain) antibody (Sigma).
'IYarlsgenic animals expressing this construct are generated ss follows.
MunT-PmeI digestion of the expression vectors pcDNA,4lMax~IJ~'II-K20L,
and PC,DNA4~~Nf ax-UPII-K20I~, respectively, releases the L chain and the FI
chazri fragments for microitzjet;LiotL FTu-K20 transgtnic mice are generated
either by 1 ) co-ir~jecdng the L chain and the H chain fragments in a 1:1
molar
ratio, or 2) injecting the MunI-1'zael fragmexit of the co-expression vector
pcDNA4IMax-I1PII-IC20LH. DNA is purified and injected by standard
techniques, bIu-K20 transgenic goats will be generated by injecting the same
cransgeties.
t~..~.w~.
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le
Expression of the rnonoeloraal antibody K~~ c~ be achieved under the
control of the goat uxomoduhrr gene prornotex in a evkaryatic expression
vector, pcDNA4IHisMax.
To clone the goat urornodulin gene Promoter, p~ w~ petfo~~ned
using goat genomic JJNA as template, with two sots of primers designed
fxom conserved regions of human and catrle UM geste promoters, A 600 tip
fragment was obtained and sequenced, It shared 94~'o and b79o identity pith
the known sequences of boys ~ hu~~M gene promoters, respectively.
14 Several s~peciHe genamic libraries were constructed pith t~ Universal
Genome'Waiker Kit (Clontech) aid PCR cuss pecFormEd using th$ Iibcarirs as
templates with several gene-specific primers designed from the 600 tsp
fragment of the goat UM gene promoter and specific adaptor primers, A 1.5
kb fragment, which includes the b40 by piece, was obtained both from one of
15 the libraries and the goat genomic DNA. This 1.5 Kb fragnnent was'subcloned
into a promoter-less pEC~FI' to force pGrUEGFP3~ axed 5equeT'ced to its
entirety (Fig. 13) ,
In ttie alternRtive strategy the Bel'°~c uromodulin gene ~ud~8 the
promoter and 3'-elements is cloned by standard teehrl(ques and tie gone of
20 interest is fused just befare;uromodtyEin's signal peptide or using
uromcdulin's signal sequences. Ia addition, the 3' end elements of the
uromodulin structural gene including introits can be fused to the 3' end of
the
gene of the interest.
The goat Ulvl gene promoter is used in the construction of Hu-K20
25 Iighc chain arid heavy chain axFresslun cassettes. PCR is performed using
as
a template the 483 w~16 expression vector DNA (courtesy of Dr. Zhou,
hTexia Biotechnologies L1c~~ trist harbors 2 copies of the chicken ~i-giobin
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-29-
insulator sequences in a head to tail orientation (Chung. J. H. et al., Proc.
Nail. Acad 94: 5'75-SBO,1997) with a S' sense primer
(5'GCc3CAGr.~.GCGGCCGC~rGTAGA.CTCG3'; sP.~~ No: ~
containing a MW site (underlined) and a 3' antiset~o pcirner
(5'CTCGCAG~'TCi~AGGTCGACaCCCCATCCTCAC3'; SEQ ID NO: d)
containing a Xhol site (underlined) (Chm'ig~ r~ H- et at., P~roc, Natl. ~l cad
Sci.
9~1; 5?S-580,1997). The ampl'ificd 2.4 kb ir~sulatorfragm~~nt of the chicken
p-globin gene is digested with Mural and ~1'iol, and ligated at the Xltol site
wirh a 1.SICb-long ~1-~f~&~'~t of the goat T~'M gene promoter,
released from FGUEG~3. ~~ PoAHA4l.1-llsMax eYp~tessiorr vector
(Invitrogen) is digested with Muni and BamHi, and this CM'S promoter-less
nectar was ligated with the Mural-BamHI eot~nbin,rd fragment of the insulator
and the goat UM promoter, 'PCR is performed using the excised'Miinl-
g~ fragment containing tha SP163 seguences (C~orman, C. M. eral.,
Pr-o~ ~Varl. Accu1 Sct. 79: 6777-6781, L9$2) from pcl7NA4IHisMa.'~ es the
template with a 5' sense primer
{5'GCCT,ATS~~.GCCiCAGA~GGGT'TG3'; SEQ m Nb: 7) containing a
BamHI site and a 3' entisensg primer '
(5'CrCGTATT~C~GGTTTCGGAGGGCGTCCG3'; SEQ ID NO: 8)
2U containing a Bswil site. Ttze PCR product is digested with BaznHi and
Bswil.
The amplified SFlfi3 fragment is dexived from the 5' untrarislated region
(UTR) of th9 vascular endothelial growth factor (VEGF) gene and it has been
spawn to increase expression levels two-to-five fold above those seen with
promoter along (Gorman, C. M. et af., Proc, Nato Acid Scz: 'l9: b77?-6781,
2S 1982) . A 320 by fragment of the variable region of the mAb Hu-l~~o lzg~
chain module is opined by J?CR amplification of pSVhyg-HuVKIC2~-HuCK
p;asmid DNA (Paul, M.A. et al., Mvz IrnmunoL 3z:14I-116,1995) ~cvith a 5'
r~. ' . . ~~ I
CA 02343104 2001-03-16
22/02 'O1 THU 13:12 [TX/RX NO 9283]
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v a tr, ~ ~ il'5f.1a37E171-I a .. a
.. . ' . . . . r. . .
~~~r
SeI~98 pr~~
(5'GCGTA CACC~AT~QATGGf~GCZ'GrTATCATC3'; SEQ1D
N0: 9) containing a Bswl s9te (underl'm~) ~ ~ Kozalc sequence (italic9) and
the start codon (bold) followed by a partial mouse V4? Ig heavy chain signal
S sequence, and a 3' antisense p~~r
(5'GCCi~TAT~YACTTACGT~GATCTCCAG3'; SEQ ~ NO:10)
containing a Hpal site (underlined). The arztplifiedproduct is digebted with
Bswxl anc'l ~p~. Anatlner Pit is performed iu pSVl~yg-HuV~K20~HuCI~.
pith a 5' sense primer
14 (5'CGG"fATGT'~Ar~GAGTAGACTfAAACACCATCCTG'xTTCG'3: SEQ
rD N0:11) containing a ~ipat site (underlined), a splice donor signal (bold)
for the ~' end of the Variable region of the light chain, and partial 5'
sequezu:e
of the hmnan ge~.4n'sC rg kappa constane region, and a 3' sntisense Per
(5'GCCITAT GAGTAGTTGGTA~'AC~~G3'; S~Q In NO:
15 1,2) containing a Prnel site (underlined) and partial d' seq~renceg of the
human
genomic Ig kappa constant region 'Z'he p~ ~°d~t ~ ~g~~ with Hpal
and Ptnel. The,three arnplifiEd PCR products ~ca iigated together through
BswI aad ~Ipal Sites with the resulting fragme~ having Barni~ ~ Prae7
e~, Tag jdin~ pGR productvrich overhangs of RamHL site at a' and Pme.I
20 site at 3' is Ligated to pcDN'A4IHislvIalInSUiator-UM whlch is digested
with
PamHI and Prner, yielding pcDNA4~l~l~xlCTM-K20L CFiB~ 1U).
For the conatru~etion of the hiu-I~20 heav~r chain expression cassette a
36~ by fragment of the variable region of the Hu-T~20 heavy chain module
can be obtained by PGR amplification of PSVgpt-VHK?0-HuCYI p~A
25 (~'aul,
M.A, et al. , Mol. Irrr.~tr~rtol. 32:101-116,1 X95) with a 5' serLSe pximer
(5'
GC(3TA C CACCATGGGATGdA~GTATCATCS'; SEQ ID
-~a . . , , ' :, .
CA 02343104 2001-03-16
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.,~~. .
-31.
N0:13) containing a ~3swX rite (underlined), the Kozak satluetice (italics)
and
the start codon (bold) followed by n partial mouse v'a~ lg heay3' chain signal
sequence, and a 3' ~tiso~e p~
(5'CiCGTA'x'y~GAGCAGACCrC3T;'rACCGTG3 ; SgQ 1D Nd:14)
containing a ~~ sr~ (underlined)- The ampli~iad product is digested with
BswiI and Clan Another PCR is performed in pSVgpt-YHK~O-HuCYI with a
5' seu5e primer
(5'C(3CT~.TATC~~.TA~TGAGTAGC'~TCTGGGGCAG3'; SEQ m N0~
15) containing a CIaI site (underlinui), a splice donor signal (bold) for the
3'
end of the variable region of the heavy ck~ain, antl partial 5~ s~ence of the
bumna genomic Ig gamma-1 ccmstant region, and a 3' antisense primer
(5'GCCi'I'ATG GACCCGCTCTGCCTCCCTCd'; SEQ ID N4:16).
containing a Pmel site underlined) arid Paul 3' sequence of the human
genomic I~, ganu'na ovrt Tegion. The PCR product is digested with CIaI
l 5 and Prnex. ?he two amplified PCR products are joined throw the CIaI site.
This joined PCR product with overhangs of ~swl site at 5' and Praol Bite ac 3'
is ligated to pcDi~IA4INiax/1JM-K2aL Whicb.is digested with Bswix andPmel
tv form PCDNA~.IMax/LTM-K20H (Fig. ll).
A single vector containing 'both !'tght chain and heavy chain modules
for co-expression can be g~ierated by inserting 'the 3~hQY ~tn~Z digested arid
5' end blunt-ceded T;3M promoter plus H cGain module of QcDNA~~~-
K20PI into the Prnel-digested pcDNa4/IVlaxlIJM 1~4L to fosn~
~D~~,~~M-KZOLH (see Fig, lx). Alternatively a bicistx~ruc
expreesioa vector can be oo~~~ted. In this case a single goat UM promoter
ZS ;~ drioing expression of both the light end heavy chains. For this purpose,
the
second UM promoter fragment in IxDNA4IMa~1'~JM-KZQL1~ can be replaced
by a fragment of an internal ribosomal entry site (iKIJS) sEquenoe ~~~' F~'
CA 02343104 2001-03-16
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~''" a:. FP, 2001='13.15 "° EPn ~d'JENCHEN +49 89 23994465 si~sea7s-ri-
. ?u'~.. ::~''s . ~. .
>~ 1 ' ra'~.~
-32-
et aL, Ccuscer Gene y'her 3; 395-351,1996),
Testing of the constructs is re>utl epithelial cell lines
Prior to the generation of transgenic arurnais using the tr~.t~sge~nic
constructs, their functionality Can be determined upon transfection in Iddney
epithelial cells and media testing for the presence of the antibody. For
example, the rabbit renal cell Iine, PAP..h~'25 (Green, N. et al., Am
J'Phyafol
249: C97-104,.1.985), oral the dog kidney cell line MDCK (ATCC irCC'L-34)
can be used to test the ftmctionality of the established expression cassettes.
The cells can be transfected with the L chiain and H chain constructs
!0 simultaneously at equal molar concentration by standard transfection
teck~nique. The co-expre~sian vector can also be transfected into tha carne
cell tines, T'werity-four hours following transfectiort, the cells'
conditioned
media can be exchanged with fresL~ media, and the cells returned to culture,
The ne~ct day, the conditioned media of the cells can be removed and applied
1S on an anti-human IgG (H chain specific)-agarose column (Sigma) for
antibody purification or tested directly for the presence of the antibody by
Western b?otting analysis. Protein concentration of eluted 'fractions is
assayed
by $radford micmassay (Bio-Rad) and fractions containing proteins can be
checked on a 10% SDS PAGE. Other functional assays for the recombinant
z0 mAb Hu-K20, for example, binding mea~rrement, T cell proliferation.
cneaawement of phosphatidic acid Synthesis, complement-dependent
cytozoxicity assay and measurement of Clq binding can also be performed.
Generation of the transgeuic animals witti the I~u-K2Q tra~enes under
the cbntrol of the goat UM gene promoter
25 Creneratian of transgenic animals cars be periormcd using standard
techxuiques
including pronucleax microirijection (Wright, C~, et aL, Bin~Technolagy 9:
CA 02343104 2001-03-16
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_- ~-r ~~;J1-~i~:l~oe ~:~ I,iUETIn~3EM +~G ~~ ?3994~E5 ~ tIF. ~'~
rl L;K ~~ t ~ ~Wi~~~7~-i ~'..~r' O
I
l
-33-
830-83,1991; Yursel, V.G. et al., .I Anlm Sci 71 Suppl 3: .1 ~9,1993; Tall,
R.J.
eC al., 27ieriogenology 5: 5 i-96$, 1996) or nuclear transfer (NT)
xr~ethodologies (Campbell, K.H, et al., tVature 380: 64-66. 1996; Wilmut, I.
er
at., .Nancre 385: 81 D-813,1997; Cibelli, J.B. er al., Science 280; I2S6-1258,
d 199$; 'Rlakay ama, T. et al., Narure 394:.369-374,1998). Munt Pmel
digestion of the expression vectors pcDNA4/A~axIUM.K2flL and
pcDNA~llvIaxJUI~-K20FI, respectively, removes the vector backbone and
generates the in.~ulator, U11~I protnater and the L chain and the H chairs
fragments for microinjection. Transgcnic animals can also be generated by
co-ir~jeating the L chain and the H chain fragments in a 1:1 molar ratio. The
transgenic animals can also be generated by i~rjjecrron of the Munl PtneT
fragment of the co~xpression vector, pcDNA4IMaxlU'M-K20LH. DNA is
purified and injected by standard techniques. .Gawrlve D1~IA of the p>iiative
transgaruc animals is prepared and analyzed by FCR and Southern blot. NT
1 S derived r~ffspring cell lines such as fetal fibroblasts can be transfected
in vitro
(Cibelli, J.E. et al., Science 280:125b-1258,1998) with the UM-KZOH and
UMi-K20L expression cassettes selected using, for exempla, Zeocin aelecuon
marker. Lines are screened for copy number and intugtation of both caesettas
prior to using them an NT experiments. Raconstnacted NT embryos can be
cultured or transferred immediately to furt4er recipient anicrtals.
Urine of trattsgenie animals can be collected daily starting at birth and
assayed to determine the quantity as well as the quality of the secreted
monoclonal antibodies.
All publications and patent applications mentioned in this specification
2S are herein incorporated by reference to the same extant as if each
independent
publication or patent application was speraficallp and individually indicated
to be incorporated by reference.
~~r
I k ,a'
CA 02343104 2001-03-16
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~~' PEA. 33J':~~i~: i' °~' EP.~ hIUENCHEN +49 89 239944b8 ,,~
~,~;~,e;~;c~m ?~: ~~ ~. _
~'~i' "' . ~"~ '
_.~'~.,.~..~~ .
-34~
white the invention h25 been described in ccmnectian vrith specific
en~bo~iirt;ents thereof, it wil! be understood that it is capable of further
modifications and this application is intended Eo co vec any variatierss.
uses, or
adgptatians of the invention following, in general, the principles of the
inv8ation and including such departures from the present disclosure that come
wiihin known or customary practice within the art to which the inventian
pertains and may be applied to the essential features hereinbefore set forth.
5,1~~~~' ~i ~~ iy ~~~n
I~ 1 E. I
' ~tN
CA 02343104 2001-03-16
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WO 00/15772 PCT/IB99/01609
1
SEQUENCE LISTING
<110> Nexia Biotechnologies, Inc.
<120> RECOMBINANT PROTEIN PRODUCTION IN URINE
USING A KIDNEY-SPECIFIC GENE PROMOTER
<130> 06632/012W02
<150> 60/100,540
<151> 1998-09-16
<160> 1
<170> FastSEQ for Windows Version 3.0
<210> 1
<211> 1547
<212> DNA
<213> Capra
<400>
1
ctcgagctcaagcttggagttgatagagctcgacattcccacctaggattgagaaaaaga60
atattaagaacttttattttcttctgaagttatagcaaagaaaggggaaaaaaaaaaaca120
ttcttatgggggataaacgggcaaaggatacaaacagttcagaaaagaataaatagtaag180
caaatgaaaagataacttcctttttcatcaaagaactgcaaaagtaaataatgataagat240
gtttctcacttttccacaaagatgaaagttaatgcccagggtggctgagtactgtgctgg300
gattgtgaactaactgttatagatctctctggggtgctgtttgggaagaaacatcgctga360
aaactgagctacctcttttcctatgaaattcccctgaggaggtgagtgagccgctgctga420
tcgtcacccgagcactaggccagacagaaggagaaagccctcaaagaggcaatgctgtgg480
atcactgtcatatttcctgctcagcctgagttcacatgtgcctgatttttctcaatatgg540
cattgccattaacgtggaattaggtcaggagacctaaggctgaaccaagccctgtcattc600
tctgccccatgactgcgcatcaccaaaacagcatcggcagtgacttccacagatggtacc660
attgctatatgccttaacttgcatcatctcctttaatggccataacaattctaggacacg720
ggtattcttgttttacagatgatgaaaattacctctggaaggaaaattactggcacacaa780
aaaacgctgaccaggattcagatagactgactccaaagtcagtctgttcatctacaaaat840
tatctacttctcaaggaccttccttcatgggaattcaaatttcttgattcacagagcatc900
tggtccaatgatgtctgaattatctgctgtctctgaccttcagccattctcagctccttt960
cctgatcacattgggaccccaggggagctggctgaatctgtgaggatgacatttgctttg1020
gaattaagtggccacaagtacacatcctggtggggacaatgagcaccccttttctcctgg1080
agcagcctggcttcagattctggcctctgcttggctccactttgtgcttttcaatgacca1140
agaaaatcccaggcccttggaattgtttactcagttaatttctaactaaagaacctcttg1200
ttgccaaaaggtataaaacagagcccttgtaactgtgggcacagctgtgacccccatgtc1260
aatcatttggggtctctacctattagggaaaagaacaacaaccacctcacagcctagaaa1320
aggaaaacactgtgtcaaaagggaaaaatattccacccccattaaaataattaagaaaca1380
gaaccagaggatcattggaggagagattgccagtgggggacagatgtatatatatagata1440
tgaaagtcacctacttgtaaaaggattaattctacctttctggtttcaggaaggtaccac1500
gtagccgaattctgcagtcgacggacggtaccgcgggcccgggatcc 1547
CA 02343104 2001-03-16
