CA2343104A1 - Recombinant protein production in urine - Google Patents

Abstract

The present invention provides methods which generate a polypeptide that is secreted into the urine by using the uromodulin gene promoter, or other promoters from genes whose products are specifically expressed in the kidney, to direct the expression of the secreted polypeptide. Also disclosed is a transgenic animal secreting a recombinant polypeptide into its urine, as well as a method for detecting such an animal while the animal is in utero. Also disclosed are methods for generating a polypeptide that is secreted into the conditioned media of cultured kidney cells transformed with a polypeptide-encoding nucleic acid sequence that is operably linked to a uromodulin gene promoter, or other kidney specific gene promoter.

Description

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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 ,_ . ,p . "y~~'1'd ,c~:~J

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- -igg$)~ ~°w~'~'~ since the bladder is not a aecretory organ, the secretion of recombinant proteins using ale uropIakin II promoters ntay be hampered.
Y~ ti The present invention provides a traz~gene that i~udes a desired ~lYpeptide whose expression! is driven by the uromodulin gene promoter, or anothor proma~s from a gene whose product is speciiicaIly e~tpressed in the kidney, thus allowing the expression arid secretion of the polypeptide from the kidney into the urine, from which it can be Isolated. ~ .
AceardingIp, lri a fit aspect, the invention provides tr~sgc~c non human urine sarcting Futimals which are capable of producing recombinant Polypeptides that are secreted extracellularly into the uci~ by the kidney ti$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.
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_3_ 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 ~ .
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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~
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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.
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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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acv. vov: . ~ M~:NCtr~:~ oe : x?- 4-,~ ~ :~6f33?f~?1-s +49 89 . . . ~' " . ~, , ~'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 ,_ 22/02 'O1 THL1 12:58 [TX/RX NO 9282) ??, FEP. ?001 13:02 EPA 1~~1UENCHEN +4g 89 23994465 ?i.. :' i0 Kl:v, vmv~'rn n~uc:~~nr.m Obi :1'/- 4-" ~' j " ,~lvGEt37f37)-, -H4y EJ;~ ' .~.
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.
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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~.
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- , _g_ 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.
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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).
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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 22/02 'O1 THL 12:58 [TX/RX NO 9282]

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-1 a-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).
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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 .. " ~ , ~ .; , G~
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a. ' ~ r 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,. ~.

22/02 'O1 THU 12:58 [TX/RX NO 92821 _%~. PEE, 2001 13:04 EPA 1~4UEN0:~IEN +49 89 23994465 NP.. :'. '_ KL Y . , vm ~ m n ~.uuulV~.ru:.m Vr1 . 1 - ~r- « : ' s ~ x4. ; 2:3of~:i7871-~ ~rø9 8 ~ ~~ ' J
-ld.
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).

22/02 'O1 THU 12:58 [TX/RX NO 9282]

%''. FF~, 20'Ol 13:05 EPA IdUEN~JHEN +~9 89 2399465 N. '_'-?3' x~v. vvw.~t-n m;rlV~.nc::v Uti :tv- 4- U ; w i~~ : ')~SC37Ei71-~ +49 E~:3 ,-av <<, ' ~ r v -t5-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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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 ..~~ie r ~, ...,.~ ~w.: .

22/02 'O1 THU 12:58 [TX/RX NO 9282]

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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~.

22/02 'O1 THU 12:58 [TX/RX NO 9282]

i~ n ' IUENn~EN +4g 89 23994465 T,~ :1,~
F..E. 2~ ~ 1 A13 : 06 ~Px 1~ .,. ,... ~ c ~LV. . ..IV1:1\vr111JY uEi . x r- y.- a : :;f'~6;3'1Ei71.-, i~h~3 F39 .i;_ ' .;.a 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 22/02 'O1 THU 12:58 fTX/RX NO 9282]

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i. ~ , - . '.~~ , 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 22/02 'O1 THU 12:58 [TX/RX NO 9282]

K«. % . FEP. 2001. 13: 06 EPh I~UENGiiEN +49 89 23994465 hr.. : l?3 .. . _ . _ :!t...._;. c76 ...- .~- ~ . ;~:s:~ ~ , U~356;~7G'T1-. ~1-4.:a d~1 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

22/02 'O:L THU 12:58 fTX/RX NO 9282]

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~~2, FED. 233. _~., .
' °' 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.

22/02 'O1 THU 12:58 (TX/RX NO 9282 "~~,., :. FEF, 2001=''1:1? ~e EF~,~dTJENCHEN +49 89 2399465 ' rIF, ''~~ ~, ,.
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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 .

22/02 'O1 THU 13:12 [TX/RX NO 9283]

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*9-9 !39 ~; n , . , -z3-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

22/02 'O1 THU 13:12 [TX/RX NO 9283]

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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 22/02 'O1 THU 13:12 [TX/RX NO 9283]

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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 22/02 'O1 THU 13:12 [TX/RX NO 9283]

K~.~_;:~. FEE;. ~~~~1,~13:' ;; u~; EPA hIUEN~~HEN +49 89 23994465 ?1:,. ' ? 9 ._ . _ , , =13r,0:~:B~lw . +4~ Bfl . , 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 22/02 'O1 THU 13:12 [TX/RX NO 9283) K~.~ w' F JF ~~,;~ it,1? ; ~ ~ ~b EFh IdUEPIC3EN +~9 89 ?399~~65 ~;b.. . ? ?~
..
. - - ' 135637(i71-.
.. , . i,~,~, ~~, , , ' 1 _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~.

22/02 'O1 THU 13:12 [TX/RX NO 9283]

~~-,.;, F,F. ?0';;1..,13:13 EFN ld'JENC-IEN +c_9 89 23994465 T,=. ,u , T " ,;,~ - , .135637671-a _.. '.:.~ ~ ., ~, . , ' ~, uL a i 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 22/02 'O1 THU 13:12 [TX/RX NO 9283]

n~a.~ ~. FOP. 2001;13:14 ~f; EPh IdUEN_CHEN +4g 89 23994465 NF. :1 i 9 ~, 9/:':.' . ~ ~ ; ~ ass:ws~z-., . +~~~ a9 ,...
;~l , 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

22/02 'O1 THU 13:12 [TX/RX NO 9283]

. ... EEP, ?331~~13:14 ~~ EPA MUEN~~HEN +49 89 23994465 '.v:, 11 r,c~ . ~.
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 . . , , ' :, .

22/02 'O1 TAL1 13:12 [TX/RX NO 9283]

FEB, ?331~~13;14~t~ EPz r~IUENCHEN +49 89 23994465 '1;36637E~'7I-. r~=. ~.'.' .,~~. .
-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~' 22/02 'O1 THU 13:12 (TX/RX NO 9283]

~''" a:. FP, 2001='13.15 "° EPn ~d'JENCHEN +49 89 23994465 si~sea7s-ri-. ?u'~.. ::~''s . ~. .
>~ 1 ' ra'~.~

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:

22/02 'O1 THU 13:12 [TX/RX NO 9283]

_- ~-r ~~;J1-~i~:l~oe ~:~ I,iUETIn~3EM +~G ~~ ?3994~E5 ~ tIF. ~'~
rl L;K ~~ t ~ ~Wi~~~7~-i ~'..~r' O
I
l 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' 22/02 'O1 THLi 13:12 [TX/RX NO 9283) ~~' 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

22/02 '01 THU 13:12 [TX/RX NO 9283]

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>

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

Claims (26)

What is claimed;

1. A nucleic acid molecule comprising (i) a sequence encoding a polypeptide, (ii) a promoter from a kidney-specific gene, said promoter operably linked to said sequence, and (iii) a leader sequence that enables secretion of said polypeptide by urine-producing cells into urine of an animal.

2. The nucleic acid molecule of claim 1, wherein the promoter from the kidney-specific gene is selected from the group consisting of a goat, sheep, pig, cow, human, and rodent,

3. The nucleic acid molecule of claim 1, wherein said kidney-specific gene is 8 uromodulin gene.

4. The nucleic acid molecule of claim 1, wherein said animal is a mammal.

5. The nucleic acid molecule of claim 1, wherein said polypeptide has dialogical activity.

6. The nucleic acid molecule of claim 1, wherein said polypeptide is soluble.

7. An animal in which the genome of cells that contribute to urine production in said animal comprising the nucleic acid molecule of claim 1.

8. The animal of claim 7, wherein said cells are kidney secretory cells.

9. The animal of claim 7, wherein said animal is selected from the group consisting of a rodent, a ruminant, human and pig.

10. The animal of claim 7, wherein said animal is a mammal.

11. A method for producing a polypeptide, said method comprising the steps of:
(a) providing a cell of an animal, said cell transfected with a nucleic acid molecule that contains (i) a nucleic acid sequence encoding a polypeptide, (ii) a kidney-specific gene promoter that directs expression of said polypeptide in said cell, and (iii) a leader sequence that causes secretion of said polypeptide by said cell;
(b) culturing said transfected cell; and (c) isolating said polypeptide from the culture medium of said cultured transfected cell.

12. The method of claim 11, wherein said promoter is a uromodulin gene promoter.

13. The method of claim 12, wherein said cell is a kidney secretory cell.

14. The method of claim 11 or 12, wherein said animal is a mammal.

15. The method of claim 11 or 12, wherein said kidney-specific gene promoter is a uromodulin gene promoter.

16. A method for producing, in the trine of a vertebrate, a recombinant protein that contains two or more subunits linked to each other by disulfide bonds, said method comprising the steps of:
(a) providing a transgenic vertebrate exhibiting urine-specific production of the recombinant protein;
(b) collecting urine from said vertebrate; and (c) isolating said protein from said urine.

17. The method of claim 16 wherein said recombinant protein is an antibody.

18. The method of claim 16 wherein said protein is a heterodimeric fertility hormone.

19. The method of claim 16 wherein said protein is collagen or fibrinogen.

20. A transgenic non-human vertebrate animal that exhibits urine-specific production of a recombinant protein that contains two or more subunits linked to each other by disulfide bonds, said protein being folded such that it is rendered biologically active.

21. The animal of claim 20, wherein the DNA encoding said protein is operatively linked to a uromodulin or uroplakin promoter.

22. The animal of claim 21, wherein the DNA encoding said protein is operatively linked to a leader sequence that enables secretion of the protein into the urine of said animal.

23. A purified nucleic acid molecule comprising a goat uromodulin gene promoter.

24. The nucleic acid molecule of claim 4, wherein said mammal is a goat.

25. The method of claim 16, wherein said protein is folded such that it is rendered biologically active.

26. The method of claim 16, wherein said protein is excreted into the urine of said animal in inactivated or partially inactivated form, said protein being activatable.