Patent Application: US-46496195-A

Abstract:
a recombinant nucleic acid construct is presented in which a mouse uroplakin - ii gene promoter is coupled to a heterologous gene of interest . when the resultant construct is introduced into a transgenic mouse , the up - ii promoter directs the expression of the heterologous gene in the urothelium of the said mouse .

Description:
two major problems of producing biologically active molecules such as protein products from cloned genes on a commercially viable scale are : ( 1 ) that bacterial expression systems frequently fail to modify the proteins properly , i . e ., by glycosylation , etc ., and ( 2 ) the subsequent isolation of gene products from the expression systems . in bacteria , yeast , and baculovirus systems the expressed proteins are most often purified from insoluble intracellular compartments . secreted proteins in yeast require specialized protease - deficient strains coupled with appropriate vectors with secretion signals . more recently , there has been success in using mammary gland - specific promoters to drive the expression of foreign proteins in these secretory glands , ultimately leading to their secretion in the resultant milk . this method has been used commercially to express human growth hormone in cows and sheep . wo 94 / 05782 . the copious volumes of milk produced by cows and sheep make this procedure attractive . however , this method suffers from several potential drawbacks : one being that the expressed protein even at relatively high levels must be purified away from a large amount of milk proteins such as caseins , immunoglobins , lactoferrins which may also entrap the desired valuable product ; another being that certain protein products may be insoluble in the calcium - rich environment of milk fluid ; and another being that this method requires the use of pregnant animals which are expensive and time consuming to produce . in the present invention , a method has been developed for expressing biologically active molecules in the luminal cavity of the bladder of transgenic mammals . urine in the bladder is of relatively high osmolality ( 50 to 1 , 000 mosmol / kg ), with ph values as low as 4 . 5 , and contains high concentrations of urea and ammonium . the lumen of the bladder may therefore provide an advantageous environment for the production of proteins that are normally difficult to express due to insolubility . the urea and high osmolality may serve as in situ denaturants and chaotropic agents . however , urine contains relatively little protein , in comparison with milk , as the kidneys are designed to prevent protein loss , therefore urothelial promoter - driven expression of proteins which by - passes the kidney produces the desired protein in a solution with relatively little contaminating host endogenous proteins . the promoter region of the uroplakin ii gene has now been elucidated . using a bovine upii cdna as a probe , a 16 - kb mouse genomic clone ( g1 ) was isolated which contains an ˜ 2 . 5 - kb transcribed region that is flanked by ˜ 3 . 5 - kb and ˜ 10 kb of 5 &# 39 ;- and 3 &# 39 ;- sequences , respectively ( see fig1 a ). alignment of the coding sequence with the upii cdna sequences of cattle ( lin et al . j . biol . chem . 1994 , 269 , 1775 - 1784 ), which are highly homologous , defined the exon / intron junctions of four introns ( fig1 b ). 5 &# 39 ;- race ( frohman et al . proc . nat &# 39 ; l acad . sci . usa 1988 , 85 , 8998 - 9002 ) experiments using mouse bladder mucosal mrna as a template established that the transcription site of the upii gene is located at 60 - bp 5 &# 39 ;- upstream of the translation initiation codon and 27 - bp downstream of a putative tata box . the 5 &# 39 ;- upstream region contains an alu - like b1 repetitive sequence (- 830 bp ) and a ( ca ) n stretch (˜- 2 . 1 kb ). finally , a polyadenylation signal resides ˜ 230 bp downstream of the translation stop codon ( see fig1 b ). the mouse upii gene is urothelium - specific like the bovine upii gene . mrnas were prepared from various mouse tissues and probed for the presence of upii sequences by reverse transcription - polymerase chain reaction ( rt - pcr ) assay . a large amount of upii product of expected size ( 266 - bp ) was generated from the bladder , but not from skin , forestomach , glandular stomach , kidney , liver , spleen , testis , or the hypothalamus / thalamus cortex and cerebellum of the brain ( see fig2 ). a rabbit antiserum previously prepared against a synthetic peptide corresponding to the n - terminal amino acid sequence elvsvvdsgsg ( 1 - 11 ) ( seq id no : 2 ) of mature bovine upii ( lin et al . j . biol . chem . 1994 , 269 , 1775 - 1784 ) immunohistochemically stains the 15 - kda bovine upii and localizes it to the superficial cell layers of bovine urothelium . this antiserum cross - reacted well with mouse upii , which contains an identical epitope , but migrates slightly slower at an apparent 17 kda mass . immunofluorescent staining of frozen sections of mouse bladders showed that the upii was associated with the all the suprabasal cell layers , suggesting that the onset of upii gene expression in mouse was earlier than that in cattle . to define the cis promoter elements for urothelial - specific expression and to demonstrate that heterologous genes can be targeted to the suprabasal urothelial cells as endogenous upii , a transgenic mouse was constructed that contains a chimeric gene in which a lacz reporter gene was under the regulation of a 3 . 6 - kb 5 &# 39 ;- flanking sequence of the mouse upii gene ( fig3 b ). the dna construct was injected into fertilized mouse eggs for transgenic mouse production . southern blot analyses of the tail dnas showed that the transgene was integrated into the genomes of 4 of 25 mice . three of these animals transmitted the reporter gene into their progeny . southern blot analyses established that the genomic dnas of these three transgenic lines , tg1 , tc2 , and tg3 , contained roughly 40 , 6 , and 30 copies , respectively , of the reporter gene per diploid genome . probing the same southern blot with the lacz sequence showed that the transgenes of all three lines were in tandem repeats and were integrated into independent sites . in all three mice lines , the transgene was expressed in the suprabasal cells of the bladder epithelium in an expression pattern similar to the endogenous upii gene . the staining correlated somewhat with gene dosage , as it was intense in tg1 ( 40 copies ) but moderate in tg2 ( 6 copies ) and tg3 ( 30 copies ). β - galactosidase activity was only observed in the bladder and other urothelia of mice that had inherited the transgene , confirming that the activity was transgene - specific . in all three transgenic mice , no β - galactosidase activity was detected in any of the non - urothelial stratified epithelia tested , including those of the skin , tongue , cornea , esophagus , and forestomach . the reporter gene product was also undetectable in all other epithelia tested , including those of liver , lung , glandular stomach , small and large intestine , uterus , and testis ; or mesenchymal tissues , including fibroblasts , endothelial cells , spleen , and various muscle cells . other urothelia closely related to the epithelium of the bladder known to cover other areas of the urinary tract , such as the renal pelvis of the kidney , the ureter , and the urethra and which also elaborate aum plaques , exhibit similar expression of the transgene . these data show that the 3 . 6 - kb 5 &# 39 ;- flanking sequence of the mouse upii gene can drive a heterologous reporter gene to express in the upper cell layers of the bladder epithelium . the lack of expression in other non - urothelial tissues indicates a high degree of tissue - specificity and demonstrates that the cis elements of this promoter region provide very tight regulatory control on tissue - specific and differentiation - dependent expression of a gene placed downstream of the promoter . as these results were corroborated in three independent transgenic lines with differing sites of transgene integration , they show that the inherent promoter activity is responsible for the tissue - specific expression and is not due to the effect of neighboring sequences of the transgene integration sites . this tight regulation is a very desirable property of any promoter used for production of foreign protein products in host transgenic animals , as it assures correct delivery to target production sites , high efficiency of expression of transduced genes , and minimizes toxic effect of aberrant expression . while these experiments were conducted using the mouse upii promoter , there is sufficient similarity between this gene in different species , so that similar results with the upii promoter sequence in other animals is expected . for example , the up gene organization ( ryan et al . mamm . genome 1993 , 4 , 656 - 661 ), cdna ( lin et al . j . biol . chem . 1994 , 269 , 1775 - 1784 ) and protein sequences , tissue patterns of expression , and morphology of aums are strikingly similar between the mouse and cow . the amino acid sequence of bovine and mouse upii are highly similar , sharing 84 of their 100 amino acid residues . wu et al . j . biol . chem . 1994 , 269 , 13716 - 13724 . in addition , although the onset of expression of the upii gene is different in these two species , upii is clearly differentiation - related in both cow and mouse bladder epithelia . in the present invention , a delivery system is provided that can specifically transform the bladder into a bioreactor capable of making a transgenic product . this delivery system comprises a transgene containing a 3 . 6 - kb 5 &# 39 ;- flanking sequence of a urothelium - specific gene , for example , the mouse uroplakin ii gene , and a gene encoding a biologically active molecule . in one embodiment , this transgene is introduced into germ cells to produce a transgenic animal capable of expressing the biologically active molecule in its bladder . as used herein , &# 34 ; biologically active molecule &# 34 ; refers to a molecule capable of causing some effect within an animal , not necessarily within the animal having the transgene . examples of such molecules include , but are not limited to , adipokinin , aldosterone , adrenocorticotropin , blood clotting factors , chorionic gonadotropin , corticoliberin , corticotropin , cystic fibrosis transmembrane conductance regulators , erythropoietin , folliberin , follitropin , glucagon gonadoliberin , gonadotropin , hypophysiotropic hormone , insulin , lipotropin , luteinizing hormone - releasing hormone , luteotropin , melanotropin , parathormone , parotin , prolactin , prolactoliberin , prolactostatin , somatoliberin , somatotropin , thyrotropin , tissue - type plasminogen activator , and vasopressin . of course , as will be obvious to one of skill in the art , the above list is not exhaustive . in addition , new genes for biologically active molecules that will function in the context of the present invention are continually being identified . the biologically active molecule can be isolated from the urine of these transgenic animals . accordingly , the present invention provides a means for isolating large amounts of biologically active molecules from the urine of transgenic animals which can be used for a variety of different purposes . in another embodiment , the transgene is carried in a vector which is well received by the epithelial cells lining the lumen of the bladder . an example of a useful vector system is the myogenic vector system ( vector therapeutics inc . houston tex .). in this embodiment , the transgene carried in the vector is introduced into the bladder of an animal in vivo . introduction of the vector can be carried by a number of different methods routine to those of skill in the art . for example , a vector of the present invention could be placed in direct contact with the urothelium via a rubber urethral catheter or foley catheter . vectors of the present invention can also be incorporated into liposomes and introduced into the animal in that form . the transgene is absorbed into one or more epithelial cells capable of expressing and secreting the biologically active molecule into the urine collecting in the bladder . it may be preferred for some biologically active molecules to also engineer a signaling sequence into the vector to insure that the molecule is secreted from the apical surface into the lumen . use of signaling sequences such as the glycophosphatidylinositol ( gpi ) linkage in anchoring molecules to a selected surface is well known in the art . the biologically active molecule is then voided from the lumen where it can be collected and separated from other components in the urine . the following nonlimiting examples are provided to further illustrate the present invention . a bovine upii cdna ( lin et al ., j . biol . chem . 1994 , 269 , 1775 - 1784 ) was used as a probe to screen a mouse embl3 - sp6a / t7 genomic library ( clontech laboratories inc . palo alto , calif .). two overlapping clones ( g1 and g2 ) were isolated ( fig1 a ) and were sequenced by the dideoxynucleotide termination method . the transcriptional initiation site was determined by sequencing three clones of 5 &# 39 ;- race ( rapid amplification of cdna ends ) products of mouse bladder cdna . a 6 - kb xhoi dna fragment of the g1 genomic clone ( fig1 a ) was subcloned in pgem7z and then restriction - cut to yield a 3 . 6 - kb dna fragment of g1 clone ( extending from the xhoi site at - 3 . 6 kb to the bamhi site at - 42 bp relative to the transcription initiation site ) and inserted into the smai site of a lacz vector , placf , ( peschon et al . proc . natl . acad . sci . usa 1987 , 84 , 5316 - 5319 ; mercer et al . neuron 1991 , 7 , 703 - 716 ) to generate pupii - lacz ( fig3 ). the 7 . 1 - kb fusion gene was excised using kpn i and hind iii , gel - purified , and microinjected into fertilized mouse eggs ( from f1 hybrids of c57bl / 6j × dba2 ), which were implanted into cd - 1 foster mothers . the lacz transgene was identified by southern blot analysis of tail dna in accordance with methods well known in the art . positive founder animals were back - crossed with ( c57bl / 6j × dba2 ) f1 hybrids to generate semizygous animals that were used for studying transgene expression . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 2 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 3963 ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( iv ) anti - sense : no ( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 11 ( b ) type : amino acid ( d ) topology : linear ( xi ) sequence description : seq id no : 2 : gluleuvalservalvalaspserglysergly1510__________________________________________________________________________