CA2134670A1

CA2134670A1 - Constitutive and inducible epidermal vector systems

Info

Publication number: CA2134670A1
Application number: CA002134670A
Authority: CA
Inventors: Dennis R. Roop; Joseph A. Rothnagel; David A. Greenhalgh
Original assignee: Individual
Current assignee: Baylor College of Medicine
Priority date: 1992-04-30
Filing date: 1993-04-28
Publication date: 1993-11-11
Also published as: AU669713B2; AU4221393A; JPH08501202A; EP0644933A1; EP0644933A4; WO1993022431A1

Abstract

A loricrin constitutive vector for efficient expression of a nucleic acid sequence in epidermal cells comprising the 5' flanking region of the loricrin gene, said flanking region containing a TATA box, a cap site and a first intron and intron/exon boundary all in appropriate sequential and positional relationship for expression of a nucleic acid cassette, a 3' flanking sequence of the loricrin gene and a linker containing a unique restriction endonuclease site at the location of the start and stop codon. Said linker connecting the 5' flanking region to the 3' flanking sequence and said linker further providing a position for inserting the cassette. The cassette contains the specific nucleic acid sequence to be expressed. Also, there is a keratin K6 inducible vector for regulating expression of a nucleic acid sequence in epidermal cells comprising the 5' flanking region of the keratin K6 gene, said flanking region including the TATA box, a cap site and the first intron and intron/exon boundary all in sequential and positional relationship for expression of a nucleic acid cassette, a 3' flanking sequence of the keratin K6 gene, and a polylinker having a plurality of restriction endonuclease sites. The polylinker connects the 5' flanking region to the 3' flanking sequence and further provides a position for insertion of the cassette. The keratin K6 and loricrin vectors can be further regulated by the addition of a Vitamin D regulatory element. The vectors can be used in a bioreactor for generating a variety of products including proteins, polypeptides or antisense RNAs. The vectors can also be used for gene therapy in treatment of a variety of diseases in animals and humans including wound healing, surgical incisions, skin ulcers, psoriasis and skin cancer, and in vaccination.

Description

.'~ W ~ 93/22431 2 1 3 ~ 6 7 0 P ~ /US93/~3993 The i~ve~tion was partially supported by a g~ant from the United 5tate~ goverr~nent u~der AR4024Q awarded by the National In~titut~s of 10Health. The ~ rernme~t ha~ certain rights ill thi~ invention.

~F~ =~
The pr~sent inve~tio~ r~a~s generally to e~pres~ion vectors for use pres~i~g protein~ a~d polypeptides ~ epidelmal cell8. More 15partic~ly it relates to a c0~8titutiv0 ve~tor COI18~tillg of the loricrin ge~e promot:er9 it~ ~' fl~i~g regio~ 19J transcribed but unt~anslated reg~o~ ~ntron7 it8 3' l;ral~scribed but u~tran~lated r~gion, it~
c~ntiguou~ non-codiIlg DN~ co~ g th~ g0Ile'8 natural tran~criptional tio:~ re~on a~d it~ 3' flallkiDg re~o~. It ~ er relates to an ~: ~ 20~ucible Yector co~ tillg of the K6 kerati~ gene promoter7 it~ 5~fl~killg regio~ 5' trallscribed but uIltra~la~d regioll, its fir t i~tron, 3' tra~c~ibed but ulltran~lated region, its eo~tiguiou~ non-coding DNA
cs~taiDi~g: the gene's nsl~l tran~c~iptio~al ~ermi~ation regio~ and it~
3' ~lank~g region. Additionally it relate~ to the trea~ent of di~ea~e 26~ing the constitutive and inducible vectors.

: i ~ The skin i~ the lar~est organ in th~ human bo~ and7 due to it~
acce~sibility, it i~ an attractive target for gene therapy. The outer layer 30of the ~kiD i~ called the epide~i~, and it i~ pa~ticular~y attractive since epidermal cells car~ be growIl ~n vitro from Ilormal and affected patients, wo~3/~243~ G70 PCl/US93/03993 are easily tran~formed geIletically by vectors, and can be readily reintroduced by autogr~ing. Pre~ious 8tudie8 investigating the feasibili~y of us~g epide~nal ceL~s for ~ene therapy haYe oIlly considered this ex uivo approach. These i~vestigation~ utiliz~d retroviTal Yector~ d their promoters to iIltroduce aIld e2~press foreign genetic material in epidermal cells. Even though the epidermis is ava~cular, these ~tudie~ demon~trated that protei~s e~pressed i~ the epide~ were abl~ to traverse the epidermal~ermal barrier alld achieve ~3y8te~c distribution (Morgan et al., Science~ Vol. 237, pp. 1476-1479, (1987), Fe~uve~ et al., PNAS USA, Vol.
86, pp. 8803-8807, (1989); Garlick et al., J. Invest. Dermatol., Vol. 97, pp.
82~829l (1991)~. The acGe~sibility of the epidermi~ mak~8 it ~uitable for other rou~s of vector d~livery that do ~ot require ~ e~ vivo approach, e.gO~ a gene gun (Sanford et al., TechIliques, Vol. 3, pp. ~16~ (1991);
William~ et al., PNAS U~A, Vol. 88, pp. 272~2730, (1991~; John~tone et : 15 : al., In Vitro Cell Dev. Biol., Vol. 27, pp. 11-14, (1991~ addition, novel v~ctor ~ystem~ derived from genes Ilormally e~pre~ed at bigh level~ i~
epide~mal cell~, could pro~e optimal for achieYi~g efficient, as well as regulated, e~pre~sioll of e~oge~ou~ DNA. ~hese vector ~y~tem~ are the ubject of thi~ elltio~.
The epidermis is a co~tinuously regene~glti~g ~tratified ~q~nou~
epithelium. Dif~ere~tia~ epiderm~ cells are the progeny of proliferative ceL~s ~ocated in the lba~al cell layer and there i~ ~ubstaDtial evidence su~ tiIlg that the rege~eration proce~ occur~ i~ pIoliferative unit~
compo~ed of slowly ~ycli~g, ~elf-re~ewi~g ~tom cells, proliferative but non-re~ewi~g transit smplii~g cell~, and post-mitotic matur~ng epidermal cell~ versen, et al., Cell Tissue Kinet., Vol. 1, pp. 351-367, ~1968);
;~ MacKenzie, et al., Nature, Vol. 226, pp. 6~655, (1970); Chr~topher~, et al., J. I~ st. ~e~natol., Vol. 56, pp. 1~170, (1971); Potten, In Stem CelLs: Their Identification and Characterization, pp. 2()0 232, (1983);
Cotsarelis, et al., Cell, Yol. 61, pp. 1329-1337, (1990)). The maturation WC~ 93/22431 r~ 1 3 ~ 6 7 û Pcr/us93/03g93 proce~s (ts~al dif~erentiatio~ initia~Qd when epidennal cell~
~nth~aw from the cell ~ycle ~d mlgrate ~rom th~ b~al layer iIltO the 8piD01~ layer. Maturatior~ cor~ es as 8pLl10ul~ ce~ migrate into the gra~ular layer a~d te~ate~ with the formation of the stratum corneum.
5 Morphological and biochemical ~tudie~ have ~hown that terminal d~erentiatio~ occurs in 8tage8. ~Matolt~r, J. InYe~t. Dermatol., Vol. 6~, pp. 127-142, (1975)). Ker~t~ K5 and K14 are m~or product~ OI basal epidermal celLs (~Noodcock-Mitchell, et al., J. Cell Biol., Vol. 95, pp. 580-588, (~ 982)). These protein~ a~semble into 10 nm ~llPment~ (intermediate 10 filame~ts EIF]) and, ~gethsr with microtubules (tubulin) and ~: micrsfVllameIlts (ac'dn), compri~e the cgtosksleton of epidermal celLs (~ei~ert, P.M., et al., Cell, Vol. 423 pp. 411-419, (1985)). One of the earlieRt Challg~8 as~ociated with t:he commitmeIlt to di~erentiation and ~gration ito the 8pi~10U~I layer ia the i~duction of anoth~r differe~tia~ioIl~specific pair of k~ratin~ (~1 ~d K10). IF co~taining K1 d K10 replace tho~e co~taining K5 and ~14 a~ the m~or products ~f cell~ in the 8pi~10U8 layer ~Woodcock-Mitchell, et al., J. Cell Biol., Yol. 9B, :PP. ~80-588, ~1~82); ROOP, et a1., PrOC. Nat1. ACad. SCi., USA, VO1. 80, PP.
716-720, (1983~; SchWeizer~ etal, cell,VQ1. 37,PP. 1~9 170, (1~84)) The 2Q ~ ~ ~ k eratin IF formed by thes~ prstein3 as~mble into bu~dles. II1 the g a~ular laly~9 a~o~he~ high molecular weight no~-IF protein i~
~thesized, which ~ proce~ed into filaggriD, arld i~ thought l;o promote erati~ filament aggr~gatio~ d disul~d~bo~d ~o~matioIl (Dale, B~., et ;, ; al., Na~ure, 'Vol. 276, pp. 729-731, (1978); HardiDg9 C.R., et al., J. Mol.
BiO1., VO1. 170, PP. 651-673, (1983)). In the f~a1 stage Of ePiderma1 Ce11 matUratiOn, tr~nsglUtaminase Cata1YZeB the cr~li~ing Of inVolucrin and lOriCrin, bY the fO1matiO~ Of (Y-g1Ut~UnY13 lysille iBOPePtide8? intO a high1Y
sOluble COrnified e~Ve1OPe WhiCh L8 1OCated jU~t b~neath the P1a~ma membrane (RiCe and Greer1, Ce11, YO~ , PP. 417-422, (1977); Mehre1, et al., Ce11, VO1. 81, PP. 110~1112, (1990)).

WQ 93/22431 P~/US93/03993 21346iO

G~e~ or cDN~s encoding the m~uor keratins expre~sed in epidermal cells have ~ow been cloned: K5 (Lersch, et al., Mol. and Cell Biol., Vol. 8, pp. 48&493, (19883, K14 (Marchuk, et al., Proc. Natl. Acad.
Sci, US~, Vol. 8, pp. 1~0901613, 51985); Knapp, et al., J. Biol. Chem, Vol.
52~2, pp. 938-04~, (1987); ~oop, et al., Cancer Re8., Vol. 48, pp. 324~-3252, (lg88), K1 (St~inert, ~t al., J. Biol. Chem., Vol. 260, pp. 7142-7149, ~1985) and K10 (Krieg, et al., J. Biol. Chem., Vol. 260, pp. 5867-5870, (1980).
North~r~ blot analy8i~ ~nd in 8itll hybridization atudies 8ugge8t that keratin e~ K5 aIld K14 are predo~alltly transeri~ed in the 10proli~erating basal layer alld transcription of kera~in genes K1 and K10 i8 i~duc~d a~ cells migrate into the ~pinous layer (Lsrsch, et al., Mol. and Cell Biol., Vol. 8, pp. ~&493, tl98~; hnapp, et al., J. Biol. Chem, Vol.
2623 pp. ~ 45, (l987); ~oop, et al., Ca~lcer Re~., Vol. 48, pp. 3245-3252, (1988)). Ge~es e~coding rat (Haydock, et al., J. Biol. Chem.9 Vol. 261, pp.
1512520-12525, (1986)) ~d mouse (Roth~agel, et al., J. Biol. Chem., Vol.
262, pp. 16643-15648, (1~7)) filagg2in ha~e ~ow bee:ll identified and in ~ hybridizatio~ esperiments haYe confirmed that transcriptiox~ of this gene i~ reBt;ricted to the granular layer (RothIlagel, et al, J. Biol. Chem., ~: VoL 262, pp. 1~15648, (1987); Fisher, et al. J. Inve~t. DeFma~l.,.Vol.
20:88, pp. 661-664, (1987)). To diate, l~ricnn i~ the o~ly ge~e encoding a ~: ~ compo~eIlt of the cornified envelope to ~ studied at the mol~cular l~velby in ~ ybn&atio~ and tran~cript~ of thi~ ge~e are rest~cted to the granul~r lay~r ~hrel, et al., Cell, Vol. 61, pp. 1103-1112, (1990)).
S~nce t~e g~ne3 ~nc~g the ~tructured protei~ described above 25ar e e~pre~sed at ve~y high level~, i.e. their indiYidual tran~cripts r epre~ent 5-10% OI the total mes~enger RNA in epidermal eslLs, their regulato~y regions could be utilized in the con~t~ction of vector~ to direct efficient e~pressioIl of Q~ogeIlous DNA i~ epidermal cell~. In parti ular, ef~orts haYe focu~ed on the gene ellcoding loricrin, a major keratinoc~e cell envelope protein (Mehrel et al., Cell, Vol. 81, pp. 1103-11129 (1g90)).

~ wo 93/2243l PCl/U~g3/03993 213j~670 Although thi~ gene i8 normally only esprsssed in the most dif~erentiated layers of ~e epidermis, the pre8ent iIlventioIl demonstra~es that it pC8~1ib~ 0 remove 8equence~ that no~ally restrict e2~pre8~ion of the loricrin gene Ln undif~ererlti~ted cells and schieve high levels of expression 5 in undif~erentiated epidermal calls (greater than the viral promoter of SV'~O). Thu~, thi~ vector i8 constitutively e~pres~ed in epidermal cells st all di~ere~tiati~n 8tate8.
I~ additioIl to the co~titutive vector, the present invention takes advantage of ~ e~pressioll charac~eristics o~ another gene e~coding the 10 K6 keratin to constIuct an inducible vector. Tbe K6 gQne i~3 normally ~eYer espressad i~ the epide~, but it caD be illduced under byperproli~erati~ coIlditioDs such a~ wound he~g (Wei~s, et al., J. Cell Biol., Yol. 98, pp. 1397-1406, (1984); Naka~awa, et al., J. Cell Biol.1 ~ol.
103, pp. ~61a, (1986); Stol~r, et al., J. Cell Biol., Vol. 107, pp. 427-446, :~ ~ 15 (1988~ d topid applîcation of retinoic ~cid (~2~osenthal et al., J. Invest.
~ ~ D~matol., V'ol. 95, pp. 51~515, (1990)).

~ :~
An~ object of the ~e~t inveIltio~ i~ a loricrin co~tituti~e vector 20: for ef~lcie~t espg~s3io~ of Ilucleic acid ~eque~ce~ î~ epidermal cell8.
~ addi~oIlal obj~et of the pr~ reIltio~ is a keratin K6 ducible ~recbor for regulated e~ ioll of Ilucleic acid ~equences in :: epidermalcells.
~ other object of the pres~nt i~ventîon îs an in viuo method of 25 transduci~g epîde~nal cells with a co~titutiYe or iIlducible vector.
A further obiect of the pre~ent i~ tio~ a bioreactor for produci~g proteins and polyp~ptid~s.
An additional object of th~ pre~ent invention i8 an enhanced method `~ of wou~d healing or healing of 8urgical iIlcision~.

WO 93/22431 - P~/U~93/03993 ~- .
~ 1 3-~`6 -7 f~

Another object OI the present invention i8 a method of treating ~kin ulcer~.
~; ~ additi~llal object ofthe present invention i8 method oftreating p8~rla~, A further object of the present invention i8 a method of treating ~: ~ ca~cer.
Thu~3, in accompl;~ g the foregoing oyects~ there i8 provided i accorda~ce with one a~pect of the preseIlt invention, a loricriD constitutive :
vector for e~l&i~nt e~pre~sion of ~ucleic acid sequence~ i~ epidermal cell~, 10 comprisi~g a 5' fl~B region of the lor~crin gene, said fla~irlg region ix~cludi~g a T.ATA bos, a cap site and a firs~ intron and an i~tron/e~con r, all in appropriate sequelltial and po~itional relstionship for pres~ of a l~ucleic ~id ca~sette; a 3' ~anking ~equence of the loricrin g~e; a~d a lirllcer having a unique re~triction e~do~uclease ~ite at the 15 loeatioll of t}~ 8t2~ d stop codon, 8alSl linker connectiIlg the 5' flanking region ~ the 3' flanki~g ~ uence and said ~er ~urther pr~Yiding a ition for i as~g t~e ~ucleic acid cassette which iIIcludes the ~pecific wGleic acid s~quence to be e~pre~sed.
In ~pecific embodiments of the pre~ent invention, tbe loricrin : , ~
20 ~: con~titu~ vec~or h~ a 5' flf~nking regioIl of appr~mately 1.~ kb9 a~l intron of ~b Ll 1~ a~d a 3' fla~king sequence of appro~mat~ly 2.1 kb.; Ill specific embodim~nt~ of the pr~ent in~rerltion, the l~ constitutive veetor al~o includ~ a poly~ ker.
A~ bl'IlatiVe emb~diment of the pre~a~t ~enti~Q L~ a keratin K6

2~ ducible ~ector or regulated e~pr0ssion of a ~ucleic acid s~qu~nceepidermal cells, comprising a 5' fla~ g region of the k~ratin K6 gene, aid f~anking regio~ i~cluding a T~TA bos, a cap 8ite, a f~st intron and tronJexon ~:oundar~, all in sequential aIld positioIlal relationship ~or e2~pression of a nucleic acid cassette; a 3' tla~king sequellce of the k~ratin 30 K6 gene; and a poly~ kerhavi~ga plurali~ of restriction endorluclease ~:
:

. ~ W0 93/22~31 , ., Pcr/U~93/039~3 ` 2i34~7~

sites, said poly-linker coImecti~g the 5' flanking region to the 3' ~lanking se~uence and ~urther providing a poæition for in~ertion of the nucleic acid cassette which includes the specific ~ucleic acid sequence to bs e~pressed.
I~ 8peCiflC embodiment~ of ~he present inYention, the keratin K6 ~i inducible vector, 5' flankiIlg region of appro~a~ely 8.0 kb, a~ intron and intro~e2~0n bouIldary of appro~ately 056 kb ~d the 3' flar~king sequeIlce of appro~umately 1.2 l~b.
I~ the presen~ i~vention, the restriction endo~uciea~e ~ites in the ~: linker or ~ly~ ker ~e selected ~rom the group coD~isting of Cla I, Not 10 I, Xma I, Bgl II, Pac I, ~o I, Nhe I and Sfi I.
~ one embodiment ~ the present inventio~, the nucleic acid cass~tte, of the con~tit;utive or inducible V8CtOI'8, contain~ a sequence g for a proteiIl, polypeptide or antiseD~e lR~A.
pecific embodime~s of the present illvention, there i~ a ~; 15 bioreaetor eomp~ing l;ra~ducsd epide~nal cells iI~cluding either the lori~ constitutiva or k~ratin K6 inducible ~ectors. The bioreactor can pr~duc~ a ~rariel~y of compound~ ~elected Prom proteins, polypeptides, antisen~e RNA.
pecific ~mbodimen~s of the pre~e~t invention, the loricrin 20 G~stituti~ire or keratin K6 inducible vectors are used for the treatment of W~ 3, 8urgical ~nc~sions, psor~as~, 8kiD ulGe~s and ~cQr.
~- ~: The me1~od OI the present i~v~lltion ca~l also be u~d for ation ~y tran~ducirlg epidermal celb with a loric~ constitutive or kerati~ K6 iIlducible ~ctor having protein~ or polypeptides which induce 25 a~ olog~cal r~spon~e.
~: Another enibodiment of the prese~t Ln~erltion L8 the nucleotide seque~ces for the loric~ gene and loriclin con~titutive vector.
Another embodiment of the present inve~tio~ L8 the nucleotide sequence~ for the keratin K6 gerle and keratin K6 induci~le vector.

wo 93/22~31 2 1 3 ~ 6 7 ~ i Pcr/~ls93/o3ss3 ~t~

Other and further objects, feature~ and adYan~ages will be apparent from the followiDg de~cription of the presen~ly preferred embodimen~s of inve~isn which are given for tbe purpQses of di~closure wheIl taken in coD,junctioll witb the accompan~g drawings.

F~ 1 i~ a ~chematic drawing of the mouse loricrin gene alld the constit;utive epidermal v~ctor deriYsd from its regulato~r sequences.
2 shows the espression charact~ tica of the co~titutive 10 epidermal vector i~ erentiated and dif~ereIltiated epidermal cells utiliziDg a reporter ge~e e~coding chlorampheI~icol acet~yl transferase (GAT).
~ 3 show~ the e3cpressio~ characteF~tics of ~e co~3tituti~e piderm~l vector in uivo utilizi~g a repor~er g~ne e~coding E. coli galact~si~e.
Fig~ 4 demo~at~ the suppre~sio~ by Yitamin Dt of a ~ovel negatiYeregulato~yeleme~tfromthehumanK1 ker~tinge~e (HK1.NRE).
i is a ~chematic r~pre~entative of th2 co~titutive epidermal ~:
wctor w}lich can be suppressed ~y Vitamin D3 vi~ insertion of the 20 ~ HKl.NRE. ~ :
Figure 0 ~3 a ~chematic drawing of a deFivati~s OI ~he mouse K6 : l~rati~ ~ene (BC~ M~6~)-HKl).
~ gure7showsth~ ~pres~ioIlcharacteri~ticsofBC:MP~K6(A)~HK1 25E~ar~ 8 ~ a schematic drawing of the mou~e K6 keratiTl ge~e a~d ~: the propo3ed cun~tructiorl of a~ ducible epidermal ~ector from its r~ at~ sequences.
Figure ~ i~ a schematic represe~tative of the inducible epidermal vector which can be ~uppressed by YitamiIl D~ via in~e~ion of HK1.NRE.

~ WO 93/22431 ` PCr/US93/039~3 213g670 The drawing~ ~re not ~ece~sarily to ~cale, and certain features of the inY~tion may be exaggerated i~ ~cale and show~ chematic form in the interest of clsrity and concisenes~.

~_ 1~
It will be readily apparent to o~e skilled in the art that Y~g sub~tit;utio~s a~d modificatioIls may be made to the inve~tion dL~closed herein without departi~g rom ths ~cope aIld spirit of the inYention.
The term "trans~ormed" as used herei~ refer~ to the proce~s or mechai~m Qf inducing changes in the characteristics (e~pre~sed ph0I~Lol~ype) of a cèll by the mec~m of gen~ transfer whereby DNA is inl;roduced i~to a c~ll in a form where it e$pre~es a 8pecific gene product or alte2s ~pr~sio~ of e~dogenou0 ~ protucts.
:: The t~rm "~a~duction" a~ u~ed h~rei~ refera to the proce~6 of 15 i~troducL g a DNA ~ressio~ ~vector i~to a cell. VaFiou~ method~ of 1;ranstuctio~ ~e pos~ible, i~cluding micro~ection, CaPO~, lipo~ection yB080~Ue ~usio~ Lse of a gene gu~ and DN~ ~ector traIlsporter.
Th~ lgricrin con~titutive vector and th~ keratin K6 indueible vestor caD be tra~duced into the squamous epithelia e~ by any of the Yariety 20 ~ of way~ dsscribed aboY~. The t3rp4s of epith~lia cells i~cluds epide~n~, oral~ e~ophag~l, vaginal, t~acheal~ co~eal ~d otheP ~qu~o~ epithelia.
Th~y ars tra~ducsd by colltacti~g the YeGt~r with the ce~. In the prefelTed em~ent t~is includ~ g a gene gu~ or DN~ vector tra~port~r.
The tsrm "DNA vector tran~por~r" as u~ed herein refers ~o those molecule~ which bind to DNA vectors and are capable of being taken up by epidermal ceJls. DNA tra~porter is a molecular c~mpl~x capable of rloIl-covale~t billding to DNA and efficiently t~a~sportiIlg the DNA
through the cell membrane. Although not ~ece~s~y, it i~ pref~rable that 30 the tra~portQr also tIa~port the DNA through the nucle~r m~mbrane.

WO 93/22431 PCrJUS93/03993 .~
21~7 0 `~

.

The term "nucleic acid ca~sette" as used herein rePers to the genetic material of iIltere~t which can e2pre~s a protein polypeptide or RNA and which i~ capable of being incorpQrated ~to tb~ epidermai cell~. The ~ucleic acid cassett~ i8 positionally a~d sequelltially orie~ted within the 5 l~ratin ~B illduci~le vector or the loricri~ constitutive vector such that the mlcleic acid iD the ca~sette can be transcribed into RNA or antisense d, when nece~ary, tra~alated into proteins or polypeptides in the tra~formed epide~al cell~. A v~iety of proteins ~d polypeptides ean be e~pressed by the se~uence in the nucleic acid cas~stte in the 10 *aIlsformed epidermal cells. These proteins or polypeptidss which can be e~pNs~ed iIlclude ho~mGnes, growth factors, enzymes, clotti~g faGtors, apolipoproteiILs, r~cept~rs, drug~, tumor antigens, viral a~tigens, para~itic ~ and bact~ tigen~. Specific e2cample~ of these compounds iDclu~e proiIls~, i~uli~, growth hormon~, i~ulin~like growth ~ctor I, 15 : insuli~like growth factor II, insulin grow~h factor binding protein, pidermal growth factor TGF-, dermal g~owth factor PDGF, aIlgiogenesi~
ctors, e.&, acid fib obl~t growth factor, ba~ic fibrobla~t growth factor and~ angioge~in for in~t~nce, matri~ protei~ such as Iype IV collag~n, Iype nI ~Lla~e~ 18~nin ~d protei~s from ~ix al, bacterial and par~sitic , 20 ~ orga~li~ which ca~ be used to i~duce imm~ologic r~spon~e.
ge~etic materiul which i~ ~corporated illtO the epidermal cell~
g the loricrin coD~ e vector or the keratin KS i~udble vector c1udes DNA ~ot normally found in epidermal cells, DNA which i~
~ormally found in epidermal cell~ but alot expre~ed at physiolo~cal ' 25 ~li~lCa~lt levels, DNA normally ~ound in epidermal sellLs and normally e2pre~ed at physiolQgical:desired levels, arly other DNA which c~ be ;~ ~ : modifi~d ~or espression i~ epidermal celLs, and any combi~ation of the ~: ~: above.
The term "loricFin co~ti~tive vector" as u~ed herein refers to a 30 vector which can be inserted into epidermal cells and which once inserted, .... ~ w~ 93/2243~ 21 3 ~ 67 0 pcr/uss3/o3993 will espr~sa a constitutive li.e., a consta~t l~vel) of protein, polypeptide or a~ti~en~e lRNA from the nucleic acid cas~ette which i~ pa~t of the loricrin con~titutive vector. The loricrin constitutive vector is used for ef~lcient e~pre88ion of a ~ucleic acid ~equence i~ epide~nal ceLl~ and i2 compri~3ed 5 of a 5' ~anking region of the loricrin gene, ~aid fla~king region im:luding a T~TA boa~, a cap ~its a~d a ~t iIltron and an iIltro~/exon bounda~y all iIl appropriate sequential alld positiollal rslatio~hip ~or e~pression OI a nucleic acid ca~sette; a 3' fl nking ~equeIlce of the lo~c~ ge~e; and a linker haqing a u~ique restrictio~ e~donuclea~e 3i~ 8t the loeation of the 10 start aDd ~top codon, said lin~er connecti~g the 6' fl~king region to the

- 3' ~ki~g seque~ce and said ~ ker fi~rther prov~ding a po~ition for in~0rtiIlg the D.ucleic acid cassette.
T~e ~equence for the loricrill geIIe which i~ used for preparing the ; ~ - loricri~ co~stitutive vector ~ shown iD SEQ. ID No. 1. The loric~
c~tit~tiYe yectoI has a 5' fl~king re8ion comprisi:llg ~ucleotide~ 1 to 1540 of SEQ. ID. No. 1; a~ iDtron a~d iIltroD/~o~ bounda~y comprising uel~otîdes 1587 to 2677 OI SEQ. ID. No. 1, a 3~ flaIll~g region co prising ~ucleotide~ 4384 to 6530 of ~ . ~. ID. No. 1; a~d a lîIlker to ~e erted at the unique ~ ite at ~ucle~tides 270~ to 2705 of SE:Q. ID.
20 ~: No. 2. The loricrin constituti~s ~ector has ~ ~' fl~king regio~ of ppro~mately 1.~ ~, an i~tron of appro~tely 1.1 1~ a~d a 3' flaIlking wque~ce of appro~imat0b 2.1 kb. Th~ ker of the loricri~ coDatitutive ~reetor caD be a poly-linker. The poly~ ker includes a pluralit~ of re~trictio~ endonuclea~e 8ite~.
The term "kerati~ K6 illducible Yector" a~ u~ed herein ~ a vector which i8 weful for regulated ~pre~ioIl oî a nucleic acid ~equence ill ~: epidermal cell~. The keratin K6 inducible vector compri~e~ a 5' flanking regio~ OI the keratin K~ ge~e, ~aid fl~king regio~ cluding a TATA boa~, a cap ~ite, a f~t illtron and an intronle~on bou~ 11 in sequential and positional relatioIlship for the espressio~ of a nucleic acid ca~ette; a 3' , .

WO 93/22431 2 1 3 ~ 6 7 0 Pcr/Us93/03993 .'~'`!, fla~ki~g ~equence of a kerati~ K6 gene; and a poly-l~ker. The poly~ ker include~ a plurality of restrictio~ endonuclea~e ~ites, connect~ the 5' flanki~g regio~ to the 3' flanking sequence and further provides a position ~or i~3ertioIl OI the n~lcl~ic acid cassette.
The partial seque~ce for the kerati~ K6 gene which i~ u~ed for prepa~g the keratirl KG inducible vector i~ sbow~ chematic form in Figur~ 8 ~d the sequellce ia ~hown in SEQ. ID No. 3. The keratin inducible ~ector ha~ a 5' ~g regioll whi~ e~tends from a unique 5' ~o I ~ite up to ~ucleotide 360 of SEQ. ID. No. 3; a~ on a~d intro~ xon bo~da~g comprising Ilucleotides 928 to 1494 of SEQ. ID. No.
3; a 3' fla~ g region which estend~ ~rom ~ucleotide 4740 of S3~:Q. ID.
No. 3 to a unique 3' Xho I site; ~d a poly linker in~erted between uoleot;ds~ 1504 to 1509 of SEQ. Il). No. 3.
The keral;iD K6 inducl~le ~ectoF ha~ a 6' fl~king regio~ of approa;imately 8.û l~ intron alld i~t~ro~eicon bounda~y of appro~imately 0.56 kb a~d a 3' flan~ing ~eque~ce of pproacimately 1.2 ~b.
~: The restrictio~ eIldonuclea~e 8ite~ foun~ he li~k~r and poly-linker of the lori~ a~d keratiDL K6 vectors can be any re~t~iction endo~uclea~es which will allow insertion of the nuGleic acid caBsette~ In the preferred smbodiment they ~re usually salected frQm the group consi~i~g of Cla I, ~ot I, Xma I, Bgl II, Pac I7 Xho I Nhe I and Sfi I.
0~2 skilled i~ th~ art will readily recog~ize that there are a Yariet~
of way~ to ~troduce the loricri~ con8titutiY~ vector or the keratin K6 iIlducible ~ector i~to epidermal cells. The vector8 can be in~erted either zn viw or e~c vivo. The mode of insertion will, to a certain degree, dete~e the aYailable methods for the in~e~tion.
One em~ent of the pre~ent iIlvention i~cludes a bioreactor. A
bioreactor i~ compri~ed of transformed epidermal cell~ which contain the loricrin co~stitutiYe vector or cont~ the kerat~n K~ i~ducible vector.
Once the vector ~ i~serted i~ the epidermal cell~, the epide~al cells will f~ ~ WO 93/22431 2 i 3 ~ 6 7 0 PCI /US93/03993 expre~s the ~ucleic ca~set~e and produce the protein, polypeptide or a~tisense RNA of i~tere3t. This can be done either in vivo or ~: vivo.
A~y compound which ca~ be e~coded i~ d espre~sed ~y, the nucleic acid ca~ette ca~ be produced by the bioreactor.
0~0 method for ex viuo int~oduction of the loricrin coIlstitutive ~rector br the keratin K6 inducible vector into epidermal cells include~ a cotrans~ection of the vector with a selectable marker. The 3electable marker is used to select those cell~ which have become traIlsformed. The ce~ ca~ ~en be used in ally of the methods described in the present inve~tion.
OD~ ~peCiflC embodime~t OI the present i~ventio~ a method fsr the enhanced hB~ g of A wc~ d or 8urgical inci8ion. Thi~ method compr~as t~e in vivo tran~ductisn of epidermal cell~ w~th a loricri co~ tiYe ~ctor or a keratin K6 inducible vector. In either caEe, the .
ucleic acid ca~sette of said vector co~tain~ a nucleic acid ~equence for a growth f3ctor.
~ the pr~felr~d embodime~t for the treatment o wounds or 8ur~cal in~ions, a pluralit y of vectors ar8 introduced iIltO the epidermal cells. I~ t~e plurality o vectors, the ca~et~e of at least oIle v~ctor 2 ~ CODtail~8 a nucleic acid aequenc~ for aIl epid*rIaal growth factor (TGF-a)9 the ca~s~tte of at lea~t one Yector eo~tai~ a dermal growth factor PDGF), a ca~s~tte of a~ least o~2e Y~ctor contains a ~ucleic acid sequence for a ma~ protei~ to anchor the epider~ ~o the dermL3, ~d a ca~sette of ~ least one ~ctor co~tai~ a ~ucleic acid ~que~ce for an aIl~ogene~i~
:~ i 25 g~ctor. The ~equè~ce for matris prote~ns ca~ be selected ~rom a~y sequea~ ful for the a~chori~g o~ the epidermis to the dermis but are usu~ly ~elected from the group con~istiag of l~pe IV collagen, laminin, idogen, aIld ~ VII collagen. The allgiogenesi~ factor i~ usually selected from the group co~istiIIg of acid fibrobla~t grow~h factor~ basic fibroblast growth factor and aIlgiogenin. The combinstion of the vestors WO 93~22431 2 1 3 4 6 7 0 P~/US93/03993 ~ ~.

-1~

provides all of the n~ces~ary elements for quick and rapid enhancement of ~eali~g of wound~ or ~urgical inci~io~. Thi~ procedure i~ ve~r helpful i~ the c~e of phstic or r~coI~structive ~urge~r. Furthe~nore, skin ulcer~
can be tr0ated by fQll~ g ~imil~ procedurç~ as de~cribed ~or wound heali~g or 8u~gical incision. These pr3cedur2s are uu3eful in ~al~ and huma~.
In the e;c vivo approach for treating or heali~g wounds~ surgical incL~io~ and ~ldn le~ions, the vectors are first tran~duced into the ~pidermal cell~ ex vivo. The tra~sformed epidermal cells are transplanted onto the aI~imal or human ~ be treated.
Another embodiment of the pre~ent invention i8 a method for tre~ti~g psoria~is. In thi~ method, epidermal celLs are transduced in vivo with a lori~ constitutive ~ctor or a keratin K6 i~ducible ~ector. A
nu~le;c acid ca~ette i~ said Y~ctor contains a ~ucleic acid sequence for a pro~in or polypeptide ~el~cted from the grou~ GQ~i~3tiI1g of TGF-~s a ~oluble form of ~ki~ r~ceptor, and an allt~en~e R~A. ThQ ~ e receptor can bs ~elected from the group co~sisting of I~1, I~6 a~d I~8.
The anti~e~e Rl!~A seque~ce is selected from the group co~sisting of TGF-a, Il,1, I~6 axld IL~.
20 ~ ~other embodime~ of the present i~ve~ioIl there i8 a method of treatillg ~cer. This msthod comprise~ the steps of in vivo rs~sductio~ of epidermal cell~ with a loricriD con~titutive ~ector or a kerati~ K6 ~ducible vector iDto epide~al cell~. The ~ucleic acid ca~ette of either v~ctor co~taiDLs the ~ucleic acid s~quence codi~Lg i~or antisen~e RNl~ for the E6 or E7 ~sne of ~he humall papilloma virue or codiDg ~or the normal p53 protein. Although the e2~ample give~ i~ for ~kin cancer, thi~ same approach i~ ~ed for cancers occurring i~ other 8quamou9 : ~ epithelial~ gi~ce the constitutive a~d inducible vectors will al30 function in the~e ti~sue t3~peB.
.

WO 93/~2431 . P~/US93/D3993 It has been found that either the keratill K6 i~duci~le vector or the loricriIl con~titutive ~ctor can be further regulated by iIltroducing the Vitamin D rsgulatory element ill~o the vector. The Yitami~ D regulato~y eleme~ is u~ually introduced i~to the 3' fla~ g sequeIlse. In the 5 present in~e~tio~, t~e Yitamin D regulato~y eleme~t is from the human K1 keratin g2Ile. With the Vitamin D regul~ r element in the vector, the ~pres~ion of the nucleic acid ca~sette~ can be suppres~ed by Vitamin D, a commonly used substa~ce ~ ~aL~ and huma~.
~n additional embodiment of the present invention ~ a method gor 10 vacci~ation cQmpr~sing the 8tep of in viuo i~troduction of a loricrin con~titutiYe Yector into epidermal cell~. The nucleic acid cassette in the ~ectors usually codes ~or a polypeptide which induees an immunolo~cal re~po~e. ~ e~ple of thi~ i8 the ~riral capsid p~otsin from the human papilloma virus. O~e ~kill~d i~ the art ~ adily recog~ize t~at any l$ other va~t y of pr~tei~s can be used to g~nerate a ~u~ologic reaponse alld thu~ produce antibodiea for vaecinatio~.
: The followi~g asamples are of ~ered by ~Nay of illustration a~d are ot i~ ded to limit the iD~Yentio~ i~ any maImer.

~: 20 }~XA~PL~3 Although it i~ a ~or kerati~o~ cell eny~lopB protein~ lsricri~
; ~ : w~ ~ot identifiQd ulltil 1990 (Mehrel, et al., Cell, Vol. 61, pp. 1103-1112, (19~0)). The p~ sequeIlce of the loricrin protei~ wa~ deduced from the o~rerlappi~g cDNA clo~es described iIl Mehrel, id. To obtai~ the ~ull g~ne, ~he cDNA ~lo~es were u~d to scree~ EMB~3 Balb/c mouse ge~omic libra~y. The gene e~codi~g lo~ was loca~ed withiIl two Bam HI ~ragments of 3.4 anid 3.1 lsb. The coding sequ~nce withill thi~ genomic fragment ia identical to the cDNA sequences and is not i~terrupted by introns. There is, howe~er, a~ tron in the 5' non-codi~g region that i8 WO 93/22431 . PCr/US93/û3993 ~-,s~
21~4670 appro~ima~ely 1.1 kb i~ le~gth. In addition to the intron and codirlg seque~ce, there L~ appro~ateb 1.5 kb of 5' fla~king sequence and 2.1 kb of 3' flanki~g seque~ce.
E~I~E 2 e~sion ve~ ro~L~he ~nQu~e lori~rin e!ene Although all of the regulato~7 elements of the loricri~ gene have ~ot bee~ ide~tified, a functional loricrin constitutive e2~pression construct was de~ ed as ~ollow~. Briefly, polymera~e chain reaction (PCR) techIlology wa~ used to delete the loricrin codi~g re~o~, leaving the 5' and 3f ~g regiQns, 5' ~d 3i ~on codillg regioDs and the intro~
(Figure 1~. A uniqua Cla I re~trictioIl site wa~ e~ eered at the 8ta~t TG) a~d ~top (T~AA) COd3118 to allow ea~ sertio~ of B~Og~D.OU~ geIle : ~ 15 ~8e~8. To a~sess the s2~pr~ion characteristics of thii~ ~ector, a : ~ r~porter ge~e, the bacte~ gene e~codi~g chloramphel~icol acelyl fers~e SCAT), was in~er~ed into the Cla I 8ite. The e~pre~ion vector was ~nalyzedbytransie~ttran~fectionintoprimar~rmoL~e epidermal cells.
Positiv~ T, lane 1) a~d negatiYe (pA10.C~AT, lane O control vector~ were iIIcluded i~ the a~say (Figure 2~. The loricrin e~pre~sion vec~r had high activil~ rellt~ted (low Ca~ medium, la~e 3) aIld differe~tiated (high Ca~ medium, lane 4) epidermal celLs, suI p~siIlg level~
ob~ed with the 8trong promoter of the vi~3~ SV40. Thi~ result wa~
ulle~pected, si~ce preYiou~ in vivo 8tUdiB~ had demo~trated that the loricri~ gene wa~ only e~pressed at a late ~tage of epidermal erentiation (Mehrel, et al., Cell, Yol. 61, pp. 110~1112, (1990))9 and indicates tbat additional ~g sequences are requiIed to 3uppre~s loric~ expre~sion in u~ erentiated epide~mal cell~.
To a~alyze the expre~sion charact~ristis~ oî the loricrin vector in vivo, the bacterial gene encodiDg ~-galacto~id~e was in~erted into the Cla ,~ WO 93/22431 . t ! `: P~/US93/039~3 I sit~. The ~ galactosidase gene has frsquelltly be~n used as a repor~er gene to 8~e8~ targeting ~pecificit~r (MacGregor, et al., In: Methods in Molecular Biology, Vol. 7, pp. 217-235, (1991)~. Thi~ con~truct wa~
designated pM~-gal and wa~ u~ed in the product;on of transgenic mice.
5 This coDstruct was dig~ted with Apa I and subjected to preparative agarose gel elect~ophore~i~ to puri~ the pM~-gal e~pres~ion construct away ~rom plas~d sequence~ (pGEM72) which mighl; i~terfere ~th e~pr~s~ion. The ~ep~rated e~pression construct seque~ces were purified a~d recovered using N~ 45 DEAE membrane (Schleicher & Schuell).
10 3DN~ wa~ precipitated and re~uspended at 1-3 nglul. ICR outbred female mice ~Sasco~ were giverl PMS ~nd HCG to ~timulate ~uperovulation, mated to E'VB males rTacoI~ic) ~d re~ulting one cell fertilized emb~yo~ were collscted f~om t~e oviducts. DN~ wa~ micro-invected into the pronuclei ~d the emb~ro~ were ~urgically transfelTed to p~euLdopregnant recipient emale~ (th~ resul~ of ma~ g ICR female~ ectomized B~ males (Taco~ic). NoR~al g~tatio~ d birth wa~ allowed to co~tinue and st appr~nately three week~ of age the pUp8 were scresned ~or evide~ce of the t~an~gene ul3ing total genomic DNA e~racted ~om the tail.
PCR a~aly~i~ wa~ performed on the e~acted tail using oligo .
20 primer~ l3pecific f~r ~-galactosidase. ~imal~ itive for the tra~gene : ~ ~ were ~u~hsr ~Iyzed to ~es~ th0 e~pr~s~ion characteri~tics OI pMI~-gaL ~ This w~ do~le by removing part of ths ear a~ld ~cubatiIlg the ti~ue i~ a ~taining solutio~ con~g X-gal. This wa~ done by removi~g part of l;he ear and i~cubati~g the ti~sue iIl a ~taini~g solution containing X-25 gal. Igpical r~ult~ are ~een in Figure 3 where a PCR po~itive ~imal expressed high levels of ~galactosidase in the ~pidermis ~Figure 3b) while a PCR Ilegative animal ~hows ~o ~uch 8taiDi~g (Figure 3a~ indicati~g that endogenous murine ~-galactosidas~ ~ no~ e~zpre~sed at suf~lcient levels in the epidermis to cause fal~e po8itive8 in thi~ y. I~tense X-gal staining WO 93/22~31 2 1 3 ~ 6 7 0 Pcr/VS93/03g93 ,~

-~8-was detected in the basal compa~ment a~ well as the ~uprabasal, more differentiated lay~rs.

To analyz~ the e~pression characteristic~ of the loricrin vector in 5 vivo~ the bac~riP~ ge~e encodillg ~-galacto~ida~e wa~ srted into the Cla I site. Thi~ data i8 show~ i~ Figure 3. Thi~ obs~rvation indicate~ that the 102~crin e~pression vector is us~ful as a coD~titutiYe vector to direct the efficient expre~sioal of e~ogenou~ DNA i~ both ~e ~dif~erentiated ~d dif~erentiated compartme~$s of the epide~.

E:2~PLe: 3 ~:: Thia e~ample d0moIlatrates t~3at a ~oYel ~egatiYe r egulat~rg 1~ eleme~t from the humaD K1 kerati~ ge~e ~HK1.NRE) ~ able to suppre~s a heter~logou~ omot~r i~ re 3ponse to Vitami~ Ds~ The HKl .NRE i8 70 ucleo~ide~ gth(~eeFigure4). PCRtec~ulogywaswedtogenerate ~:: B~HI arld Bgl II ~ite8 at oppo~ite e~d~ of thi~ f~agme~t. This ~acilitates generati~g multiple copies of this fragme~t ~ce ligation and dige~tion 20 wit~ BamL HI aDd B~l II ~ select for oligomers which have ligated head to tail. Four ta~dem copie~ of the HKl.N~E were i~erted into the Bgl II cloni~g sit~ of pA10.CAT. In the abs~ce of Vit D~ co~truct is highly oxpre~ed Y he~ fected i~to primary mou~e epidermal c81L8 ~Figure 4). The sdditior3 of illcrea~g coIlce~t~atio~s of Vitamin D, to the 2~ cultllre m~dium ~ompletely suppre~ses tra~scription of thi~ heterologouspromoter. Thus, by usiIlg Vit~ D ,, the acti~rit y of the e~pres~ion vector is modulated. Figure 5 shows a schematic representative of a deri~rative of the loricFiD con~titutive epidex mal vector which contains the HK1.NRE
in it~ 3' fl~king region. The activit~y of this vector withiIl epidermal cells ~- " WO 93/22431 21 ~ 4 6 7 0 PCI /US93/û3993 can be suppre~sed by topical applicatioll of Yitamin D3, or an analogue, to the ~kin.

EL~llPLE 4 I~Q~ Ld-~ of a MQuse ~6 ~çr~in ~ene Se~eral laboratories have reported that kerati~ K6 L~ ~ot e2~pressed ormal epid~rmLs, but i~ e~pre~sed unde~ hyperproll~erative co~ditio~s 8UCll aB wouI~ding SWei~8, et al., J. Cell Biol., Vol. 98, pp. 1397-1406, ~1984); Naka2awa, et al., J. Cell Biol., Vol. 103, pp. 561a (1986); Stoler, et al., d. Gell Biol., Yol. 107, pp. 427~46, (1988)) or topical applica~ion of reti~oic acid (lR~senthal~ et al., J. I~YeBt. Dermatol., VO1. 957 PP. 510-515~
(1990). Although K6 eacpr~sio~ doe~ not occur i~ inter~ollicular epid~rmis, it doe~ occur in hair follicles (Nakaz~wa, et al., 3. C8ll Biol., ol. 103, pp. ~61a, (19B6)). ~ent re~ults i~dic~t~ th~ ~here are two K6 16 c3DN~s that dif~er i~ ~equellce i~ only a fsw nuc~eotid~s. These cDNA
clo~e~ ha~e beell ~d to difl~ere~tially scr~sn a E~BL 3 Balb/c mou~e omic libr~y alld isola~is two di~tulct K~ ge~eB. The~e genes are closely ked wil;hi~ genLomic DN4 i.e., a~ged in t~dem. Th~y h~ve almo~t identical 33 hal~a, inCludiDg ide~tical 3' no~oding and flanking regions.: ~ 20 ~ I~tere~ti~gly, the 5' hal~e~ ofthe 2 g~es di~er greatly ~ their IeBt~ictioIl ~a~e~t pat~r~. ~sque~ce anslysi~ of t~e ~egion ~ear the ATG ~hows ma~y dif~ere~ce~ b~twee~: the two g~lle~. The ~que~ce of olle of the~e e~s, desi~t~d BCM-~K6(0, i~ show~ ~ SEQ. ID. No. 3. To determiI1e the ~pre~io~ characteri~tic~ of t}li~ gene in viv~ in tran~gcrlic 25 mic~, PCR tech~ology wa~ u~d to modi~y a 1~.5 kb ~ho I fragment collta~ing B~M-MK6~A). Nucleotides encodi~g the ~termi~al region OI
1;h8 K~ proteill were deleted and nucleotide~ eneo~g the amins) acid sequence SEQ. ID. No. 4 w~re in~erted. These amino acids ar8 at the C-terminal of huma~ ksrati~ K1 (Joh~on, et al., PNAS, U~A, Vol. 82, pp.
1896-1900, (1985)). A ~chemstic represe~tative of thi~ derivative of the w093/2243l 213~7~ PCr/US93/03993~ ~

mou~e K6 gene (BCM-MK6(A~-HK1) i~ 3how~ i~ Figure 6. ~tisera haYe previously beerl g~ller~ed agsi~t the HK1 ~terminal peptide ~ enthal, et al., J. Inv~st. Dermatol., Vol. 95, pp. 510-515, ~1990)). The~e ar~tibodies are mono~pec~fic for this human K1 peptide a~d allow expression of the deriva$ized BCM-MK6(A~HK1 transgene to be followed against the e~pression p~ttern of the endo3e~ous mou~e K6 genes.
The d~rivati~ed tnQuse K6 transge~e show~ ~n Figure 6 wa~ used iIl the production of transgenic mice a~ outlined in E2~ample 2. Mice resultirlg from the iDitial iluectio~ were ~creened by PCR analy8i8 for pFesence of the BCM-MK6(A)-HK1 tra~ge~e. Po~itive Iounders were i~itially a~a~yzed for t~ g~ne ~cpres~ion a~ follows. A small ear biop~y w~s taken ~d after 48 ho~ a ~econd biopsy wa~ hken at the same site to scsr~ for ~2~pression during wound h~aling Tra~gene ~pres~ion wa~
limited to hair follicle~ i~ the ~itial biop~ d was Ilot preaent in 1~ ollicular ~pidermis. TraIIsgene e~pre~sion was obs~rved ~ the epidenni~ in th~ 48 hsur biopaie~, but ollly at the 8ite of woundi~g. To further conf~ the illdueibilit~y ofthe BCM-MK~(~)-HK1 transgene under hyperproli~erative conditiolls, F1 gensration off~riIlg from the initial ounders were treated topically with the hypeIplaEiogenic ag~nt 12-0-: ~ 20 : tetradecaDoylphorbol-13-seetate. Bispsie~ were take~be~ore a~d 48 hours ; ~ ~ ~r topical applicatio~ OI this agent. Immunofluorescence wa~ p~rformed : ~ on ~ozerl ~ection~ of these bispsies with anti~era specific ~or the H~1 peptide. No e~pre~io~ was obser~ed pIior to the inductio~ of hyperplasia, however, the BCM-MK6~A)-HK1 proteiIl was e~pres~ed at very high level~
iD all layers of the epidermi~ 48 hours aPcer hyperpla~ia was induced (Figure 7).

.

NO 93/22431 2 1 3 ~ 6 7 0 Pcr/US93/03993 E~A~PLE ~

R~sults obtained wath the derivatiYe of BCM-MK6(A~ (Figure 7) 6 indic~ th~t all of the regulato~y ~equences required to ~uppre~s ~gpre~ion of this gena i~ normal epidermis alld activate it~ e~pression under ~yperprolif~ratiYe coIlditions, such a~ in wou~diIlg hesling or ~erim~tally iIlduced hyperplssia, ar8 located wnthin the 13.5 1~ Xho I
fra~e~ (Figure 6). Ther~fore, an inducible Yector was de~reloped from 10 this fragment. Thi~ vector is ver~ useful in gene therapy applications w~ere do~age o p~rmac~uti~ need~ to be re~a~d. In addition9 thi~
ector i~ ideally ~uited for ~Nound healing applica~ion~ ~ince it is i~d~ced dur~g ~he wound h~ g pro~ but ~uppre~ed ~er heali~g ha~
~cu~ed. Figa~r~ 8 illwt;rates how a vector ~ ~tructed f~om the BCM-: M~6(0 g~lle. The ~ctor i~ deli~red from th~ 13.5 kb Xho I fragment which ~co~ta;m~ the l!Dti~ K6 ~e~e. Th~ ~ame g~eral 0tra~gy u~ed in ~: : cox~l;~uction of the co~tituti~.re epidermal vector (Figur~ 1~ is follQwed.
Th~ expres~ioI~ ~ctor:r@tains all of the 6' flanki~g ~equencQ~, the 5' non-::
CodiDg ~quenees up to but not includiDg the AT&, ~he ~lrst intlon 2() ~clu~g the ~plice-~ite~ OI tb~ intron-e3co~ bounda~ d all of the 3' on-codiDg aDd fl~g sequeIlce~ afl;er the T~A codon. A polyli~ker i~
~eered 3' of the f~ i~troIl to allow ~aE y i~ertio~ oî esogen~s DNA
.
ea~et~. Th~se m~ipulations are peaformed through the use of PCR
tech~olo~ ~ique Xho I ~ are co~r~ed at the e~d~ of the vector to 25 allc~w ea~y a2nplifiQtion in pGE~ ~ectors aIld e~ci~io~ for purification fi om pla~mid ~equ~nc~8. Rece~ in viuo re~ts indicate that the ~: endoge~o~ humaIl ~6 ge~ inducible a~er topical application of all-tr~ retinoic acid. Further, in vivo mouse espe~me~ ,uggest that the vector ~hown in Figure 9 is inducible by topical application OI retinoic 30 acid, or a~ analogue, ~o the 8ki~.
.

W0 93/22431 213 ~ 6 7 0 1~Cl/US93/(~3993 E~ea though the inducible epidermal vector depicted in Figure 8 i~
5 ~uppre~ed or ~ilellt in normal epide~, i$ ca~ be accidently illduced ~
11 F91~Ult of iIU~. ThereIore, it i~ desirable to have an additional ~uppres~or eIlgineered i~to this const~uct. In addition, thi~ 8uppre880r i8 used to more tightly regulate pharmaceutical delive~r. Thi~ is achieved by insertion of the HK1.NRE described in F'igure 4. Figure 9 shows a 10 ~chematic repre3e~tatiYe of a derivative of the K6 iDducible epidermal vector which co~tai~s th~ ~IK1.NRE ~ its 3' fl~ki~g region. The acti~n~ of this vecto~ with~ epidermal cell~ uppres~ed by topical applicatioIl OI VitamiD~ D~ or ~ snalogue, to the ~kin.

E~AII~L~6 7 ;~ ~
Greater tha~ 3.5 ~illio~ indiaidual~ develop skin ulcers. Duri~g ormal he~i~g, epidermal cells p~uce gr~th ac~r~ which a~fect ~ot o~y epide~ cells but al~o cells ~snthin the dermis. In addition, 2û epidermal cel~ the~ize several ma~i2c protei~ which pro~ride an ~chor to the underlyi~g derm~. Many ~ ulcers occur in patients with diso~r~ such as circulato~ly proble~ aIld ~betes, ~d the normal healiDg proce~ in impaired. Th~ ducibl~ epidermal vec~r is u~ed to targ~t the combi~ pre~ion of grow~h factor~, to accelera~e growth of 25 cells in both the epidermal alld dermal compa~meat~; xnatri~ proteir~, to incre~ teD~ile ~trength; and angiogene~ ac~o~, to improve circulatiorl, in aIl attempt to impro~e healing these patient~.

WO 93~22431 2 1 3 ~ 6 7 0 P~fUS93/03993 in ~n~ thera~y a~pro~che~ ~o can~çr 5kin ca~cer L~ by ~ar the mo~t commo~ form of u~cer with greater 5 than 600,~ ~ew case~ reported each year. SeYeral ~ne8 have been implicated in causiIlg BkiIl cancer, iIlcluding 1088 or mutation of the host tumor 8uppre880r gene~ p 53 and e~pre~sion of the E6 arld E7 forming gene8 of human papilloma virus (HPV). In uitro studies ~ugge~t tllat the normal or ~d t~e p53 gene can reYert the pheno~pe 10 of malig~ant cells or illduce programmed cell death. The constitutive epidermal vector is used to target espres 3ion of the Ilormal p53 gene to cau8e reveraio~ to a rlon-~aligna~t phenol ype or i~du~tion of ~rogrammed death in vi2Jo. I~l c~cers where HPV i~ ~a~ected OI being th~ etiological t, the coitutive vactor i~ used to target e~pression of anti~snse NA ~pecific ~or the E6: a~d E7 genes of HP~.

2ûP80~;B iB~a: conlmo~ herited 8kiD di~ s which affect~
pp =ately 4 millio~ i~dividual~ in th~ U.S., 20 million world~wide. It charsctsriz~d by the presQnce of inflamed 8caly ~3kin. Although ~e pecific def~ct for p~oria~ not known, illappropriate e2pression of growth fsctor~, and ~ytokine~ ap~ to be r e~ sible for it~
~:; 25 ~pathoge~e~i~. Epidermal vectors are u~ed to inhibit the mitogenic e~fect~
of positiYe growth gactor~ produced ill p~oriatic le~ion~ by ~cpr~8s~ng nega~ive growth factor~ which induce growth arrest of epidermal cells.
The inflammation ob~erved in p~oria~i~ m~st likely re~ults from :~ ~nappropriate e~pre~sion of ~ytokines. Targeted e2~pres~ion of ~oluble 30c~ ne receptors prevents st~nulation of an illflammato~ ltrate in .

W~ 93t22431 . PCr/VS93/03993 ~
~3 1~

-2~

thi~ disea~e. In aIlo~her approach, aIltisense RNA i8 directed again~t tran~ript~ of positive grow~h factors or ~ytol~nes. The~e approache~ haYe therapeutic pote~tial for other dermato~es resulting ~rom i~iflammation.
All patent~ and publications me~tioned in this specification are 5 andicati~e OI the 10vels of tho~e s~lled i~ the art to w~ich the invention pertains. All pate~ d publicatio~s which are incorporated herein by re~re~ce are incorporated to the same e~t a~ if each indindual publication was ~pecifically and i~dividually indicated to be incorporated by refer~ce.
C)Ile skilled irl the art will readily appreciate that the present e~tion i~ well adapted to c~r out the objects and obtain the ends ~d advaDtag~ ~entio~ed, a~ well a~ tho~e inhersllt thereiIl. The bioreactor~, n~leic acid ~qUe~Ce8, tra~formed epidermal cell~, loricrin constitutive ~ctor a~d keratin K~ inducible vector, along ~th the methods,; : 15 procedures~ treat~e~t8, mol~Gule8 o~ ~pecific compoundl~ described her~in pre~ently repre8elltatiYe of preferred embodiment~, are e~emp1ary and are ~ot intended a~ limit~tio~ on the scope of the invelltioIl. Changes thsreill and other u~s Y~ occur to those ~killed in the art which are e~compaased ~nthin the spi~it of the iIlventio~ as def~ed by the scope of : 20 ~ the claims.

::~

.

... . . . . . ..

f,~ WO 93~22431 PCr/US93il)3993 ` i 21-34670 ,25.

~lSQ~ I8~ING~

( 1~ GEN}S~ INFO~SATION s ( i) APPl.ICA21Ts ~oop, l~n~is R.
Rothn~g~l, Jo~ph A.
Greenhalgh, David A.

( i~ ) TITLE OF INV~NTION s C:ONSTITUTIVE AND INDUCI8LE BPIDE:RMAL
V~CTOR SYSTE~S

L ~ 1 ) NUI~E:~ OF S~QU~i:NC~S s 4 lv ~ COR}~SPONW5NC15 ~DD~SS s (A~ ~DD~SS8151:s Fulbrls~ht ~ J~wor~ki ~B) S~US~s 1301 ~oglnnoy, Sult0 5100 s I C ) C~TY s Hous~on~
(D) sr~ 0x~
20~ : CO~Y- ~U.S~.A.
) ZXP~ ~701003095 A) ~SæDIlJ~ s ;~ Floppy di~k (B) CO~UTE:R~ PC eo~patible :~
C~ OPISRATING SYS$~: ~C-DOS~ DOS
5D) 80rrliARE) P~t-tltIn ~l~a-e ~I.û, V9~rl3t~0n i~

T APPLICiU~ON DA~A~
30 ~ (A) I~PP~ TIO~ R~
~ 8 ) F~LING DATlI~
(C~ CLA88Ir~ r~t :: ~v1 ii) A~ Y/AG15NT INFO~ION:
: ~ 35 ~ A ) NA~: P~u l, Thom~ D .
; (B) ~aECISTRATION NU~BlSR: 32,714 ~ ~ ~: (C) R~ NC35lDOCÆT ~ 2iUMB~SR~ D--5dOS

:~ :
, WO 93/2243 1 PCl /US93/03993 .~
FZ 13.~G7 0 .

( ix ) TEL~SC~M}~UNI~:ATION INEORMATION: ' (A) TEI~:PHON~t 713/651-5325 (B) Tl~L~FAX: 713/651-52~6 (C~ ISX- 762829 2 ) INFOR~ATION ~OR SEQ ID NO s 1:

~1 ) 8EQ~!:NCX CHARACTERISTICS s (A) ~:N~;T~t 6530 ba3e pair~
~B) TYPEs nuclelc ncld ( C ) S~R~DEDNESS s double (D) TOE'OLOGYs l~n~ar ~:

rp~: s D~A ( gsnom ( ~ll ) ~YPOI ~S~TC:AL s NO
: .
AN~ NSE 3 NO
:20 ~ xl )~ 8BQU151~C:15 DESCRIPTION s SEQ ID ~O ~1:

25 GGATCC~ AT~G~GTCT ~GAGAC TATCCCGGGG C:AGC~AC AC~GAAGTGS~ 60 ATe~c~a TCA~;6GA~rG GGTGA~TCAC AGGGCCCCCA ATGT~AGC T~GAGA~GA 120 -:
ACCChGGG;~G = AT C:ACCTCAGTT CATACTGTCC AAP.CTGAAAC AAGTGGCAC)~ 180 AGTTTCTCA~: ~CA~Tt~ ATC:AGGP:r CX:TTTAGATC Al'TWaTGCTe CCCC~TAATT 240 P~:A~T CTGATT~G~h T2ATTCmC AACACJ~GCTG G~;q~GGAAC~A GGTTCI~ACAG 300 35 TGGTA'rCT7~A ATM~C:AACTG AGTTCCA~TG ATS;AAAt:AAA GGA~CAC TATGTTCTTC 360 ATAC:ACAGAG GGGGGGT~:T CTTGGCCCTA GGGTCATCA& AG~CTGAGT ~TCTTATA 420 GGAAAATAGT ~ GATGTCT TcAcAcAc~r c~rccAAT AGG&~C~AG GGC~GGCATG 480 ~ . WO 93/22431 2 1 3 4 6 7 0 PCl/US93/03993 ATT~:AAGG~ AA~Gq~GTTCT GTCATGTGAG AA.AAGAGQA AAGTA~AAT AT~AC~TACT 540 ATGTAGTACI~ TTS:ATAmC I~TAAC~CCA Tl~C~ATGTT TCTGTGAA~T AAATTATAGG 600 5 ATTCC:TC;CTT GGTAG:A~CAA A~ATCA GACI~GCTCAA CAATGAACAA GTAGTC~GTA 660 ACTGCCC~GT TGGTGaC~TT GCA~AACTA CTGTGCTTTG CCCATG~;TGA CATAS;CTTGA 720 A~T~T~ATG GAAGACCTGA ACCCAACTaA GATCTC:TAAG TAS~TTCCAC TCTATGGTGG 780 CATCTC~aAG GTCAGAGTCA ~GTt;CAGCG CCATAGGACA TCAGI~ATCAA AGGGTCATGG 840 TGAAAAGGCT accAGGGTcT GTC7~GTTAG ~CTCACC~T TGTAAGTAAA GTI::AGTAGTC 900 15 AC;TI~ACI~AG ATCAAAACAC CTGCTCTQC AI~GGAAT~C TTAAAGl'AGA C~AAAGTt:AT 960 ~CTAGTTACA GTGCTGrCST T~CCG~ A CCA~CCQAA CTGGG;F~GC$G GGGACTCACG 1020 AAC~ CA ACt~ G T2~A~;CA&A~C AGh~GS:AACC C~ATGAAGTG ~ Ac 1080 A~TG~C ATJUU:Aa~Ta aATTCTAAAA m~GAGAAT ~CC~GATA 1140 ~GAAATTAA AACCAI~ T ~AAAATTGGA AA~ATACAAC Tt;A~C$AGCT TC~.TGTC~T 1200 25 AGACAATGTe ~T~GATCq~CT AGATTCCCTA l~GGC~GCTTC A~:TCTGC AACCTAGTCC 1260 .

~CTI~G~ .G C~ C~G5~ aTG~cA~cA ACC~ ACAG AAGm~TGAi~ AACAATq~TCT 1329 .
GCCATCCAC~ CCA T~rCTAaTGA CCAAC~Gt:T CACTGl~ACA TU~GAGaAGT 1380 a:C,C~GTC~T ~C~C~A~T CCCTA~CCt:T ATCCCA~GAA l~;lUA~CT TCA~GAATGG 1440 GTC~ATCCTT CCCCTGCAAT CAC~ GG AGGT&CCTGA TU~aTAGAT~: AGTC~GAGCA lS00 35 ~;ACAAGAGT ATAAAACAC:A GGAGCACCAG TGTCCCTCAC AT~Gt:~TCA l:C:TCCTTCCC 156C

TC~CTCATCT TCCCT&GTCC l~C~ Tl~G TGTWC:CTCT CC~;GCTGTC TGGTCTCTCC 1620 A5TTGGCCT~ GCTCAGCTTG CAGAGAW~ AAS;GAACAGA GCCTTTCTCC CCTTTGG;A~G 1680 WO 93/22431 P~/US9~/03993 lii7~

GTACTCTGr~ CA~Ar~GAGA AGGGCT~TAG GAAAGCACTG GGAGAGTGGT ~AGC$GGTGC 1740 TGG~AGATG ATGTGT~TGG TCTTCTGGGC AGAATGTTAA AACTTCACAA AGATATGACT 1800 ~TC~C~TACT ~CTCTGGC~C CCTGGGAGCT GA~GGTTAGA ATACTGGATG ACTGCAGTGG 1860 CAGGCCTCGA TGGGC~&GAT GAAGCTTTTG AAC~TGCCAG AAGTGGCTGA ATACACTATC 1920 A~GAAGGGAG ~GGGACGATA ~GTCA~AGA~ ~GGTGCTGAT GGGAGAT~TG AGAAGCCACA 1980 AAAACCCAAG t T(:~;~T~ TGAGG~5;CAGA TGTTCTGACA GATAAATGAC TT~TG:AGGTG 2040 CTGA.ACTACA CAGCTTCCTA TTAGCTACl~G CTAATTGGAG TCTACCAAAT TTAGACTCCT 2100 15 GCAT~T~CA AAA.aGP.TGTC TACTq~CTTC q~;GTTAGATG TACT~TC:CA AAAGGTTCAG 2160 AGTq~iCq~CCA ~GTTTGCA GAU~GGACC~ QGTAGAGCT GTC:TTGTC~A A~TTG(;CC 2220 ~ AGGAT ~T~CACTC~ ATAGGACa~A TCAAGAGm AAP~ AAGGA CTTTATACAG 2280 G~GCTaA q~TCCAAACa AATe:T~mCT TATTGTGCTG GGAaT~:GATA AAATS:CACGT 2340 ~TT5~:; CAA~TTCTA CTCAAT'rTAA AGAATC~GCA ~G~GACTTÇ; GGAGCACCC:T 2400 25 'r/40AC~a8A GTGTTTArr'rA ATGTAAGATC AAAAGC~GT 6GGAATGTGG Gt;GTTCTGCT 2460 ~ccc~aaTc A t:~TAGTAGAA GAAAGGCAGA C'rTGAGGCA~ A~GGGGGTU CrATTAACGG 2 5 2 0 C:ACS~lGAA GAGCTAACC~a GTCC~G~;AAT GCAGTCQ&3~ CTAaTCT GCAT~AGCT 2580 3~
A~A~TCA~;A AG~ATG~GG C~TGGATGCA TCTGCC~CCT T~:AQGI::GTC CTCTTGCTGC 2640 TGTTGGTCq~A A'rGTTGCTCr TCTG~TC TTC:CAGGGl~ CCC(:TTCTCC TTi~AACAAGA 2700 35 TGTC5'CACCA GAAA.AAGC:I~G CCCACTCCCT GCCCTCCTGT WGTTGTGGA AAGACCTCTG 2760 GTGGAG~;AG~; AGGCG~:CGGC GGCTATTATA Ga~GTGGCGG CTCTGGCTGC GGAt:GC&GCT 2820 C~TCTGGAGG AGGCTC~AGC TGTGGAGGCG GAGGCGG~GG TTCCTATS:GA GGTGGTTCCA 2E~S0 .~ WO 93J22431 PCr/US93~03993 2134~

~9 GCTGC~:CGG TGGAt~ C TCCGGTGG&G GCGTCA~GTA CTCCGGAG~:C GGCG~TGGCT 2940 C~AGCT~GG CGGCG~:~AC TCCGGAGGCG GTG&TGGC~C TA5:CTGCGGC GGTGGS~TACT 3000 5 CT~;G;GGGCGG CO~CC AGC9~ G GTGGCTACTC c~aG~cGGc ~GCGGCTCCA 3060 GC~ CCGCGG CGGCAGCTAC TCCGGGGGSG GCTCCAGCI~G TGGAGGCGGT GGCU:t:TCTG 3120 GTGGGGGCGT CAAGTACTCC G4AGGTGGTG GCSGOG&CGG CTCTAGCTGC oGCG4CGGCT 3180 CCTCCGGGGG CGGC~GCGGC G4C~CC~GCT GCGGAGGCGG ATCAGGAGGC GGCGGCTCCT 3240 ACTG~GGAGG CTCCTCTGGA GoCGaC~GCT CCGGTGGC~G OGGC~GC&GT ~CCG&AGGCG 3300 15 GCA~G~ C TCGTo6CG&C GG~GGC~C~A GCTGCWAGG CG5CTATTCC GGC~GCGG~G 3360 GAAGC~G~ CGGCTC:TA5C T~:TGGOt;GO~ &~AC:TCA~;G TGGCGG~Gt;A TCQt;C~ G 3420 G =Ct;~ B~A~ C GCSG~CO GCAt~ GCTG C:GG~GGTG:GT ~CCT~:CGGTG 3480 ~20 CGG~TCeTCC C~ACAG~ I!C AGTt;CC~GAG CTACGGA~aC GG~TG~ ~C:G 3540 G~G~TCCAG CT&CGti:C~GG &~;CTI'.Cq~CCt; GGGGCGG~GB ~CCAGCTGC GGTGGCGGCT 3600 25 A~CCGGGGG CGGAGGCTC~ A~G Ga:GCTCCTC Tl;G~GGTGGC TCCAt:TTGCG 3660 t:~;G :G~:CI~C ~Aq~CCGGT0 G~CGO~SGG ~GCTGC~;C CCCGGCTCCT 3720 C~ A~;C¢GGCT~T q~ T~ TC AGCAGACQG ~G;ACCTCt: TGCGC:CCCCC 3780 A~GAGC~A CGGA5aGG~ TCTTCC~CaAt; GAGGTGGTAG ~:TGS;AG&T GGCTCt:T~G 3840 GCGGCWT&G C~;GCGGTGGC q~ACq~CCA GOWTWTGG C~;GC2~GCAGC GGTGGCTGC& 3900 35 ~TGGAGGCTA ~CC~GAaGC GBCGC~GGCT Gq~GGC^GCGG ~CTTCCGGG GGCAGCGGCG 3960 GTG5CTGU:C: AGGTGCT TCC(;GACGC:A GCGGCG5;TGÇ: CTGCGGAGGA GGCTACTI:CG 4020 GAGGCGG~GG C:GGTGGCTCC AGCTGCGGAG GCGGCTCC:~C TGGTY:GCGGC TCT~:GAGC;TG 4080 W O 93/22431 PCT~US93/03993 .~ ~
~13~671~

GC`AA ~ TGT GCCAG~CT~C CACCAG~CCC AGCAGAAGCA GGCGCCTACC TGGCCGTGCA 4140 A~TAAGGTCA CCGGGTTGCA ACGGAGA~AA CAGAGCTGGA AGAGT~CTCC GTGGGCGCCG 4200 ATGGGCTTAA CI~TC~rG A~Tr5GCCIC AGaTTTCCAA ACCCTTCACA TTTTAAGCGC 4260 CCCTTCCCCC ~A~AAGCC ATTGAGTCGC TCAAGGTGTA TCCT~TTCTG CAGATTTTTC 4320 ATCTTGGTTT CTG~ATG~CT ACCTCCC~AT TCTAGTGTCT CCT Q GTCA~ TAAATTTGCT 4380 ATTCATGAGA ATCTCT~AGT TTG~TGTAGT CTTTGTAGCT TGCAAATTTA CTCAGTTCAT 4440 TCTGTGTTTG CTrTTTCCAT TC~TT~GTTC A Q ~TTAAAT TCAC~GAACA AGTGTTCTAT 4500 CCCAA ~ ~GG GGGAGSAGAT AGATGGAATG GGGCAAAGGA TGAC~AAGGT ~GTG~ACAGT 4560 ~ ~GTC GCTTAAA~A~ ~TG~G~TG4 TC~TCAAACA CCAAGAAA~G TCSTCAC~GG 4620 ACATCC~AC~ ChTCA~aaA AT~GGaCCTG O~C~GGCAAT TTC~AGCAGT ~CAGA~TTCA 4680 : ~TCTCCh~OT T~W ~AGCA &GATGGCTC~ CaGATTA&ÇT SAGCTACC~G ~GGTCCAAGT ~740 CC~C~5~ ~ S ~T~GACCr AAGAAGA~G ACATTCA~CC CTGAA~AAAA GAC CCTGCC 4800 CA~GC~ATCT ~CCGGA~C~C TA~AACTACT TTCCT~ACTC AT~ACCCATG ATAGAGCTTT 4860 GAGGC~aGA ~AU~CCCT CTAT~;TCTTC SCAAGI~ C ~G~TCTTC:A TTAAS;CC:TGA ~3920 ~AC~TT~TTA CCaGOGCACG TCTCC~CAAT ACTGATAAAG TCTGG~TTTG TTAGTCTGTT 4980 ~
AG~q~AT 'rATAq~C~GA~ AATCAAGATC CTCTACAGTG TGTGAGAC:AG TTTACTGAt:;C 5040 ATCTATAG~G ATAGAA~GCA oCCCTCr5GA ~GGAT~aAAC GCGTAC~TrT OGTCC~A~TT 5100 3~ GAGAAGG~AC ATCGTAAGTA T~TAAGATGC TTAACATCAG T~TCACAGAG GTCACT~4AA 5160 ACATTAGGG4 CCTCCT~ATT AGCAAGCATA AAGCTAGAGT TGCT~AAAGG CATGTG~AC 5220 A~CCATCCCC TGGCCA~ATC CTGTTTTACA GTCAGATSTT ATGAGCrTSA GGTAAATGCT 5280 ~ WO 93/22431 P~/US93/03993 ; ~ 213~670 AACTTACSGA ~TTACTC~AG TTA~ m TGC ~ATACTAAhA AGC~AATGTG CC~TCCTACA 5340 TTTACCTA~T GATAGAAATA AAA~GATTTC ATCTCACTCT TCCATTTGGA GTCATCACTA 5400 5 CCT~CATCAT TTGC~CA~A GA~AGAGCAT GCCAAGTAGC AACCTCAGTG ACACAGTAGT 5460 CTTACCACCA C~TTTTTATG GAT~AAAT~T ATI~5TTIIA GCATGGTTAT ATGTGCATAT 5520 ~ ATAcAeTcT GA~TACTCAC TTCCCTA~CC mcTT~Tc CTCCCCATCC CAACCTGTAT 5580 CAA~C~TTAC CTTCCCTACA A ~CCCTTT~ CCA~GTTTTT G~TAGTT~TG TT~GTTTGTT 5640 TTGTGACCC~ CTGA&CTAAC CAGGGC QTC TGTATGACCA ~GG&TTTGGA TTCTGATGGA 5700 ATCCCAGTGG GTACACAACT GAAACT~T~ ACTCCCCTTC ACAGAA~CT~ TCAGTAGACA 5760 As~arrcaAc AGG~A~T&OT G&5GCTCT~T CCaTCCT5GG CTAA~GTT~ AC~&GA~AGT 5820 C~TGTGC~ ; CC~C~GQG ACAACCATAG l~GCTGTGAG CTCATGTTTG CAATGGCTGT 5880 20 ~ ~
GTI~lITACl~TA GG~GATA~TA ~l~GGACCC ATTI~TCQTG rcTGGcT~ ATATTCCACC 5940 TTC~CTmA a;~ TCCT TGA~:T = AGGAAT&m TGGTT~GAI~C CGAGTGCTQ 6000 :
2~ GT~GTCl m I~TTCAGAA TCT~GAGCAT QA~GGATAC ATA~GATATT ATATT~TAGG 6060 ~: ATAC~T ~q~CAGA TT~TT~TAI ACCCTTCI~TA TTG&TTAACC ATAATCCCCA 6120 :~T~TSr~CT CCTCTAAC~C TCCACT¢CTC CCaT~CC~GA TGAAAGCTT~ CAA~TCCATG 6180 ~
TATTTTCCCT CSITGCIITC ATTT~ATC~A TAI$GTATGA TCTCA~CTCC CTTAATC~AT 6240 CT~SA~ Q ATAACCCTT~ TCTAAhClGG TAGCC~ACAA ~TTTA~TCC AGT~CTTGAT 6300 35 ~C~GAAGTA~ ATGGAGGAAT GT~AACTCAT GCTQGCCTG GTCTATGGAA TGGGT~CAA 6360 ~C QGCC~GG ~CTA~a~AAT AGGACCC~GT C$CAAAAACA ACTAAACCAA ACAAACAAAC fi420 ' WO 93/22431 . PCl/US93~03993 .~
213~670 AAACAAAGAA U~ACAaACA AaQAAcc~ AAATCTCAAC ChTTTCTAG~ TTTTCTAGTT 6480 TTTACTT~U~ CATC~AGTTA AGCA~AAC5~A AAGTTTQ~A AATAGGATCC 6530 ~ 2 ) INFORM~T~ON F~R SEQ ID t~Os 2:

( i) SEQU3:NClS C~ARACTERISTICS:
(A) I,P~NGTH: 5092 ba~a palr~
~) TYP}I s nuclei~ ~Icld 0 ~ C ~ STRA~ DNESS s double ~D~ TOPOI~GY: llrl~r 50LECUE~5 TYPE: DNA ( genomic ) ~ ili ) HYK~rHETIC~l s NO

( lv) AN2I--SENSlI: NO

~ 20 ~xl) SlCQUE~aOE WÇ8C~XPTIONs SEU ID NOs2:

GGA2CC~r aTAGCTI:T~r~Crl~IGAGAC TATCCCt~GGG C:~raGCA~AC ACAGAAGTGG 60 : ~5 ~rGCTCA~G S~GGGATTG GGTS;AATC~C AGGGCCCCCA ATGl~rGGA~c TAGAGAA~GA 120 .
A,CCO~G _ CACCTGAGT~ CTG~CC AA~CTGA~C AAGTGGCi~CA 180 A¢mCTC~U; AGCC~AACTC ~ CA¢GAT CGm~GATC Aq~ TC CCCCATAATT 240 AAGAC~T~CT ~rGAl~CAG~A ~A~C:rrTC l~ACAQG~G BG~GG~AC~A GG~TCil,ACaG 300 TGGT~TCTTA ~TAGCAACq~G AF~CU~ATG ATGA~A~aAAA GGAAAAAC~C TASGTT:::TTC 360 35 ATACACAGAG GGGGGCTGCT CTTGGCCCTA GGGTCATCAG AGA~AcTe~AGT AAP.TCTTATA 420 ~;AAAATAGT TAAGATGTcr TCACAC~CCT CCTTTCU~AT AGGGTTQAG GGCA~GCATG 480 ATTGGAAG~A A~AGTGrTCT GTCATGTGAG AAAAGAGCAA AAGTATTAAT ATCACATACT 540 Aq~GTAG`rACA TTCATATTTC ATAACTTCCA TTTT QTGT~ TCTGTGAAAT AAP~TTATAGG 600 ATTCCTGC~T ~GTAGACCAA ATGGGGATG~ G~CAGCTCAA CAATGAACA~ GTACTCAGTA 660 ACT~CCCTGT TGG~G~C~TT GCATGAACTA C$GTGCTT~G CCCATGGTGA Q~AGCTTGA 720 ~ATAGTAATG GAAGACCT~A ACCCAACTG~ GATC$CTAAG TACATTC Q C TCTATGGTGG 780 CATCTCAG~G GTCAGAGTQ CTG$GCAGCG CCA~AGGA~ TC~aAAT~ A~;GTCATGG f340 TG ~ GGCT GC QGGGTCT GTCTTG~G TTCTCACCTT ~GTAAGTA~ GTCAGTAGTC 990 : AGTAACAAAG ATGhAAACAC CTG~TCTCAC AAGGAATAAC rTAAAGTAGA CTAAAGTCAT 960 GCT~r5ACA GTGCTGTCT~ TTCCGTG~$A CQTCCCAAA CIGGGAGC~G G&GAC~GACG 1020 CTC~CA ACCAATA~a~ T~AGCAGA~C ABA~GC~CC CAArGAAGTG ~SCATGAAAC 1080 ~ GTGGC ~A.AGAG~TG GAq~T&q~AAAA TTTTGAG~AT l~CCAAGATA 1140~: 20 A50AA~5~AA A~AAAGAT CAAAaTSGGA ~AGA~CAAC T~A~CT~G~T TCT~TCTT 1200 hGaC~ArGTC T~AGATCTC~ ~GAIICC&TA AGGCTGCTTC ACAAGT~TGC ~ACCTAGTCC 12~0 :
~ 25 ~GT~5A~5~G CC~TCTGGTT ~T~CACGCA ACCTATACAG AAGTTTT&AA ~ACAATTTCT 1320 ~ ' ~
: ~ GCCATC~G~ C2G~GG~CA T~C~AA~GA CCA~CCTGC~ C~C~GrTAC~ TCAGAGAAGT 1380 ~ :CCAGT~A~ ACACCAAACT GCCSA~CCC~ A~CCCI~AGAA mG~AT~ TC~TG~TGG 1440 ~ T ~ TCCT~ CCCC5oCAA~ CACAGGCAGG ~&GTGCCTGA ~CAATAGATG AGT~GAGC~ lSoo ~GAC~GAGT A~aAAA QC~ GCAGC~C QG TGTCCCT QC ATCAGCATCA CCrCCTTCCC 1560 TCA~TCATCT TCCCTGGT~C T~CAGG~AG TGTGGGCTCT CC~GGCTGTC TGGTCTC~CC 1620 AGTTGGCCTT GCTC~GCrTG GAGAGAGGTT AAGGAACAGA GC~T~SC~CC CC~TT~GAAG 1680 GTACTCTGTT CAAATTGAGA A~GGCTTTAG GAAAGCACTG GGAGAGTGGT ~AGCTGGTGC 1740 W O 93/22431 PC~/US93/03993 213~70 ~

TG&GCAGATG ATGTGTCTG4 TCTTCTGGGC AGAATGTTAA AACTTCACAA AGATATGACT 1800 ATCTCCTACT TCTCTGGCAC C~TG&GAGCT GAGGGTTAGA ATACTGGATG ACTGCAGTGG 1860 ~AGGCCTC Q T&GGC~GGAT &AACCTTTTG AACCTGCQG A~GTGGCTGA ATACACTATC 1920 AGGA~GGAG Ao&GACGAT~ AGTCATAGAA TGGTGCTCAT ~GGAGATTTG AGAAGCCACA 1980 AA~CC~A~G CTCTGCrTT~ ~GAGG&CAGA SGTTCTGACA GATAAA~BAC ~TGTGAGGTG 2040 C~GAACTACA CAGCTTCCTA TTAGCTACAG CTAATTGGAG TCTACCAAAT TTAGACTCCT 2100 GCAT~TCT~A AAAAGATGTC TACTTTCTTC TGGTTAGATG TACTGGTCCA AAAGGTTCAG 2160 ~GrTC5$CCA ~ rTTG*A GACAGGACC~ C~GTAG~GCT GTCTTGTCTA ATAATTGGCC 2220 ~S~GGAGGAT ATCTCAC~A A~A05AGAGA TCAAGAG m AAAC~GGA GTrTATACAG 2280 GAA~Cr~A ~aTCCAA~Q ~CrrTTCT TaTTG~GCTG CGAGTGG~A AAATCC~CGT 2340 ~C~A~XST~ CAACrT~C~A C~aATr~A AGAA~CAGGA CTGGGACrTG GGAGCACCCT 2400 r~oaca~Ki~ G~GmATTA ~TGTAAGATC ~ GC~G&T GGGAATGTGG ~GG~TCTBCT 2460 2~ TCCCrA~SCA C~T~GTAGAA CAA~GGC~GA GTTGAGGGAA AACCCGCTC~ CTA~TAACGG 2520 ~ACTr5rG~A GAGC~AACCA GTCCAGGAh~ C&AGTCCAGA CACCT~GT~T GCATAAAGCT 2580 .
AGG~GT~aGA ~GTATGS5B4 CAT~ATGCA TC5GCCACCT ~CAC~CG~C CTCT~GCTGC 2640 5~TTGGTCTA AT~T5X~:T~ TC~GC$~TTC ~TCC~&&ST~ CCCCTTCTCC T~AAACAACA 2700 TCGA~AAGGT CACCGGGTTG CAACG5AGAC AAC~GAGCIG GAA&AGTTCT CCG~GGGCGC 2760 ~¢ATG&GC~T A~CTTTC~CA TGAAT2TCCC TCAGGrTTCC AAACCCTT~A CATTTTAAGC 2820 GC GC~TCCC CC~GAAGAA~ CCATTGAGTC GCT ~ GG~G TATCCTGTTC TGCAGATTTT 2880 T~ATCS~GGT T~ClGhATCA CTACCTCCCA ATTCTA~TGT CTC~TCAGTC AATAAATTTG 2940 WO 93/22431 21 3 ~ 6 7 0 PCr/US93/03993 CTATTCATGA GAATCq~C~GA GTTTGCTGTA GTCTTTGTAG CTTGCAAATT TACTCAGTTC 3000 ATTCTGTGTT TGCTTTSTCC A~CATTAGT T~C~TTTAA ATTCACTCA~ CAAGTGTTCT 3060 ATCCCA~GGT G~;GGAGTAG ATAGATGGAA TGGGGGAAAG GATGACCAPG GTTGTGAACA 3120 GTCTGGGGTG TGBCTIAAAA ATCATGa~:AT GGTC5~TCI~AA CaCQAGAAA AGTCTTCACT 3180 GGA~TC~A C~CATC:A~ AAa~CC TGC~;QGGCA Al~TCTAGC~ GTGC:AGAGTT 324t) QCTCq~CCAA GTTCTGGI~G CAGGATG5;CT CTC~GATTAG GTTAGCTACC AGAGG~CCAA 3300 GTCCACTGAC ATGTTCTSi:AC CTAA~ GAA GGAC:ATTCAC CC~::TGAACAA AAGACCCCTG 3360 15 C:CC~TGCGAT CTTCCGCAAC ACT~TAACTA ~TTCCTTAC TC~TGACCCA TGa~TAGAGCT 3420 TT~AGGCA~A GaTACAAACC C'rCT~TBTCT TCrCAAG~l'T GCCAGTTCTT CATTAAGCCT 3480 ~ a~rAccTTc~ TAC:CAGCl;CA CGTCTCC~GA A~ACTGAT~A AGTCq~GGTTT T~TTAGTCTG 3549 TT~Ga~aAA5! ATTAq~TCAG~ ATAI~CAAGA TCCSCTACAC TGS~;TÇ;AGAC AGTTTACTGA 3600 CCATC~ AGATAl;AA~:G CAGCCCTCTT CAI~GGATTGA ACGCaq~ACGT TTC~;TCC:IUT 3660 ~: 26 TTGAGAA&~T AC~TCt;~AG TATTTA~GAT ÇCTTA~CATC AGTATC:ACAG AG&TCACTGG 37i!0 AAACA~ CC~GCTGA T~3~G~ TAl~ GA GT~ A GGCATGTGTA 3'780 P,CAP.C~TCC C~ GA TCCTGmTA CA~;T3~AGATT TTATCAGCTT TAGGTAAATG 3840 l:TAAC~T~Cq~ GACq~C~CA AG~ ~ aCTATACTAA AAAGCCAATG TGCCTTCCTA 3900 CATTTAGCTA ATGATAG:AAA SAAAP~AGATT TCATCTCACT CTTCt:ATTTG GAGTCATCAC 3960 35 TAi::CTTCATC AT~TGCA$CA GAGATAG:A(:C ATC;CQAGTA GC~CCTCI!~G TGACACAG'rA 4020 GTCT~ACCAC CACATTTTTA TCGATTAAAT GTA~ TAGCATGGTT ATATGTGCAT 4080 ATAATACACT CTGATTAC:TC ACTTC:CCTAT CCTTTCrrAC TCCTCCCCAT CCCAACCTGT 4140 W O 93/22431 . PCT/US93tO3~3~
21346~U ,.

AT~AATCCTT AC~TTCGCTA C~A~CCCTT ~ACCA~G~TT TTGTTAGTTT TGTTGGTTTG 4200 TTTTGTGACC CA~TGAGCTA ACQGGGCCA TCTGTATGAC CATGGGTTTG GATT~TGATG 4260 GA~TCCCACT GGG~ACACAA CTOAAACTAG T~ACTCCCCT TCAQGAATC TATCAGTAGA 4320 C~TAATT&A A~AGGGAATG GTGGG~CTCT CTCCATCC~T ~GCTAACTGT TGAC~GSACA 4380 a~cTTGTGcA GGCC~AaTGC ACACAACCA~ AGTT&CTGTG AGCTCATGTT TGCAAT~GCT 4440 GTGT~TaCA TAGGAOA~G TATTTTGGAG CCATTATCCA TGTCToGCTC TTATATTCCA 4500 CCT~CTCTTT T~GaATGTTC CTTGAGTCTT TGAGGAATGT IITGGTTA~A ACCGAGTGCT 4560 15 GACTTGTCAT rTATTTTCAG AaTCrTGAGC ATCAAA5GAT A~ATAA~ATA TTATATTATA 4620 W ~ TA A T5~TTG~A Q GA~ITTTCAT ATACC~TTC~ TATTGGr~A CCAT~ATCCC 4680 ChA5rSSSC~ CSCCTC~AAC AC~CCA~TGC TCCCA~ACQ G~T6AAACCT TTChACTC~ 4740 ~0 TG~A~TI~CC C~C~IloC~T ~CA~r5TATC T~AITGTA~ C~CAACT CCCTT~ATCT 4800 ATCT ~CTAC CAA~CCCT ~TTCTAAACT GGTAGCCTAC ~ACTr~AGTT CCAGTACTTG 4860 ATGCAGAAGT A~ATÇGAGCA A~TGAAC~C ATGC$CAGCC T~GTCTATGG AASGGGTTAC 4920 : ' AAGCCAG~CC oG~TATaT~ ATAGGACCCT GTCTCAAAAA C~A~TAAACC AAACAAACAA 4980 ACAAACAaa~ RACAAhCAAA:CAAAGAAACC AAAAATCTQ ACC~TTTCTa GTTTTTC~AG 5043 ~5SSS~CTSO AACa~C~AG~ TAAGCA~AC ~AAAGTTTQ AAAATAGGAT ec 5092 (2) INFORMATION FO~ S~Q ID NO:3: :

~i) SEQU~N OE CHARACTERIST~CS:
(A) LR~GTH: 5l59 b~0e pair~
(B) TYPEs nucleic acld (C) STRANDEDN~SSs doubl~
SD) ~OPOLOaYs lin-ar WO 93/22431 2 1 3 4 ~ 7 P~/US~3/03993 ( ii ) ~OLi3~CVL~ TYPE ~ DNA ( g~nom~ c ) HYPO~:TICAL: NO

~ iv ) ANTI--5ENSE ~ NO

(xi) SEQUENCIS i:~SCRlPTION~ SEQ ID NO-3:
GG41~AAAt:CT GTGTGGTGAG GGGGC~ GGGAGTGTCT ACATGGGGCA A~AAGGAAAG 60 GG~C~TT~T CAC~CA~C~G CTCCTTGTC:T CT~GTTTG AGAAGATGAC TAACTCATGA 120 15 CTTAl~ GAA T~rTACGTCCT GGCTCATTGT s;TTClWATcA A&TCAAG~t:T ~;AAGGU~GG 180 M;~ATTTGCT CC~TaAC~ a aGc~Tcc~ A1~GCAATCTT C~;TATCAT ACC:TTT~AG240 ~M~ TcAT~r A~G~AAAG CCCTGt:Cf:TA C:CCACTCTGC AAGCTCACC:~ 300 2~ :
~CIU:aACC~:a ACCC~ . CA TCTGT~CCAT aq~TA~ÇCGB CTGCS~ GAt: C5r~CACAC 369 TCAtCl'CsS~ AaCTCTG~::CC ~GCCGT~CT CTA~:TTCt:Q GCCTTCTCAT C~Ct::AGG~C 420 2S CATGTC~ACC ~AAACCACCA T~ GTCA AACC~GCCAC CGTGGCTACA GrGCC:AGCTC480 ; .

~a~AG~G C~GC$CA A~:CGCq~GG ~TCI~GQG~ GTGTCCG~GT GCCGCTCCCG 540 , ~;aGcA~;c~t;~ ~GC2CC~C;TI; CAI~Tt;~GTGG aGGAGCTGGC TTTGGCAGCA GGAGCCTC:TA 600 TGG:TGTG&a~ AÇ;CTCCa~CJ~ .T~CCAT CGG~G ~ C:AGCTGSeGCA TTGGAGGAGG 660 ~TATGGCAGC C~ATTTGGAG CAA~CTTOGG CATTG5AWT GGAG~TGGT~ GTGGCTT~GG 720 35 CTT~GG~GGT G4A~C~G~CT T~G~TGGTGG CTAT&G&GGA GCTGGCTTCC CGGTGTGCCC 780 ACC~GGAGGC ATCCAAGA~G TC~CCA~CAA CC~GAGCCTC CT~A~CCCC TG~ACCTGCA 840 ..

~AT~GACCCC ACCATC~AGC GGGTGAGGAC TGAGGAGAGG GAGC~GATCA AGACCCTCAA 900 W O 93/22431 . PCT/US93/03993 ~.
~134670 TAACAAG~TT GGCTCCrTCA TCGACA~GGT GAGACATGGT CCTCCCTAGA GCACCCTGTG 960 TGTCT~CAGG GAATGCTGAA ~AGAGGT&TA GGGAAGAGGC TTCAGTCTCA GCTCTG~TAC 1020 TGCCTGTGTT GCTAGTTGAT GCTCTGTCCT GGTTTGTGTT CCTCTT Q GT TAG~CTGGCA 1080 CCAGTGGACT GAeATEACTT AAAGACTCAC AAA~CAGGCT TGTAGGGA~ TGGAAGATTA 1200 T~AFTATGTA T~GTGCAGTT GGGAGGCA~G C QGCCTCAC TAAGCTGCAG CACACTTCAT 1260 15 CATTGGG~GC TTCAG~CTGG ACTGTr~CCC TCA~ACCGA& GGTCAGGGTC TAACTACACT 1380 : ~ :
~ GA~G~CS~T AGT~aG~CAG CC5~A5AG&G TACACACACT AG$B~AG~GT ~T~GAAGG 1440 ; :
~ ~GAAACC~A AAC~CTCCC CCTCaTACTT GCCCCCCOGC CCCCACCAG& TGCGGrTCCT 1500 ; GG CAGCAG AACDAGGTGC TG~ACACo~a G~GGGCC~IQ ~GGaAG~GC AGGGcaccAA 1560 ; ~ ~ GaCC~iCAGC CAGAACC~GG AGCC~ArGTT TGAGCAGTAC AT Q G~AACC TCCGCAGACA 1620 : 25: GCTGG~GC ATCATTG!t;AG AGAGGG4TCG: CCTGGACTC~ ~AGCTGAGGA aCATGCAGGA 1680 cAcaG~GG~a GACT~CAAGA GChAG~6AGT ~ACAAAGAAG GGAGAATCCA 6TCTCC~GAC 1740 : :TTTATkAA~A ~GoAAGCCCA AAT~TAAA~A ~G4GCTCCAT GATGTAAGAA AGCTTG~TCA 1800 ~30 ~A~C~G~5AC AGAGGCTGCC ~TTGA~ACCA TCCACCCCGT GGC$CCAATA T~GTG Q CCT 1860 ~TCCTCTTGT AGATATGAAC ~TGAAAT~AA CAAGCGCACA GC~GCAGAGA ~5G~AT~CGT 1920 G~CCCTGAAG AAGG~GAGTT GACTA~CCAC AAGGATGGGT TTCTCTG~GG ~A~GACATAA 1980 AAGGCCTTGT ATA~CTGCGT CATTCC~GAG AAAT&GTGGT TACAGGGAAA G~AGTGAACG 2040 GTCTGS&GAA GAGAGGTAAC CTGAr~CCAT GTTCTTGATG GTTTTCTCAG GATGTAGATG 2100 ~ ~ WO 93/22431 2 I 3 g 6 7 0 PCr/US93/03993 CTY;CCTACAT GAACAAAG~ GAl~CTGCA~G CCAAGGQGA CAGl~CTAAQ GATGATATCA 2160 ACTTCTTGAG AGCTC~CTAT GAAGCAGTAA GCCCCCCT~G TCTT~CTTC TCC~TTCt:AT 2220 5 ~CACCACTCC CT~TA~TT TSCCCCC~GG GCAAAGTt;TT TGACCTCTC;C A&TTCT~A 2280 S;ACAAAGATG ACTATGGCTC TTTCTG'rCCT GC~G4AI~CTG TCTCAGATGC AAACTCACAT 2340 CTQGAQCA TCTGTGGTCC TCTCC~TGGA CAACAACCGT AGCCTG~;AC:C TGGACAGCAT 2400 CATCGCTGAG ~TCAAGGCCC AGTA~GAGGA CATTGCT QG AGAAGTCG&G CTGAAGCTGA 2460 ~TCCTGGTAC CAGACTAAAG TaAGTATTGG GGTGG~GGCT GATGGGGATG CCTGGGGTCC 2520 ACCCTGAACT C~A~GAGTCT CTG~GTTCAG ~Ar5GGAGGC CCACT~A~AG AAAT~GGGAT 2580 .
~ ~ GTT~TCCGAG AhAAIGCACT GTGCACATGT ACC~TAGAAT AATGm~c TCGAAGAGTA 2640 : ~AAGA~CAo~ G~GG~AGAT& GAAAGITGCC ATAAATGG W TCCATGCTCT T~GCTTGAGC 2700 2~ :
aA l~ TGa~q~CC T~:AAA Çq~GAGAACA Tq~ TC CTG~GGG~ C 2760 TATGGAGTCT GTGG~ CC~ AAAoCT~TC T5GAGGAA~A GCCAG~AC~T CCATG~AAGT 2820 : 26 GT~GCCACT ~AGAG~GGG TTr~GT~ccG CATGT~ACAA CTC~CATAGA TaTCCTCTCT 2880 T~GATTGoC~ T~CAG~ATGA GGAGCTGCa~ OTCA~AGC~G GCAG~CA$GG GGACGACCTG 2940 C~CAAC~CCA AGCAG~AaAT ~GCT~AGA~C AACCGCATGA TCCAG~GGCT GAGATCTGAG 3000 AT~GACCACC TTAAGAAGC~ GGTGGGG~AG ~CAGAGAAAT GC~TGGG$~G C~GG~TGTGT 3060 TTCCIGTCCT CTAACT~TTG CTCACCAGA~ ~CCATGGTCT GGGGCTCAGC CTCTG~GAG 3120 3~ ~TGTAGACTC Q CGArTATT TTTGTTGC~C TCTC~GCCCA GTGTGCCAAC CTGCAAGCTG 3180 CTAT$GCTGA TGC~GAGGAA C~TGGGCAGA ~GGCCCTGAA GGA~GCC~GG ~GCAAGCTGG 3240 AAG&GCTGCA GGATGCCCTG CAGAAGGCCA AA~AGGA QT GGC QGGCTG CTGAAGGAGT 3300 WO ~3/22431 . PCr/US93/039~3 ~
2~34~7a `

~o-ACCAGGAACT C~TG~TGTC A~GCTGGCCC TGf;ATGT~ GA AATTGCCACC TACAGGAAGC 3360 TGC'rGGA~; AGAGGAGTY;C ~GGTGGt;T~A CTATATCCTC CAACCCCTS:A GGACAGCS~CC :~420 5 TTGGT~;CAAG CAC~ GCAC A~aGAAGGGAC CACTGACTAT GCCCACAAT~ GTCt:CTTTAA 3480 GA~ACTCCTT GCTGTGCTGG AGAGATGGCT CATTGTTTAA GAGCACTAAC TCCI'CTTCCA 3540 GAGTTACTGA GTTTAATTCC Qt~CAaCCAC ~GGTGATTC ACAATCATCT CTATTGAGAT 3600 CC~t;TGCCCC CTTCTGGTGT GTTTGAAAAC AGCTACAGTG A~CTAI~AATA CATATACTAA 3660 ~TAAAGAATA TTTTTAAACA AACAAACAAA ACAAAACAAA CAAAC~AACA ATC~ACCCAA 3720 C ~AGTGGATTC TCTCTGAGCC TTCACTAGA'r TGAGGCTTCC CATTC:AGGCT 3'7RO

GAAG~GA~; ~GCC~t;T~ CTCACC~G~T GCTTTCCTCT Tt;TAGGrrGA AT~;GTGAAG~; 3840 ~ G~;ACCA 6TCl~Cl~ GTAAG~ACTC TG~TS;TCCG AATCCCCTTC T C~TACTTT 3900 GT~;GC;~AT ~ATCTGGTCA ~;TGOGC~G AC~TG'rCTG q~GS;;TGTCCl~ T~CCTCC~C 3960 ACAGCTGTGG ~AGTCCAC COTGTCQGC G4C~ CA GTGC~;G& TGCCAGCPGC 4020 :25 AGCTTAG~CC TGGGTGGAGG Qt~ CTAC TCCTATAGU~ GCAGCCATt;G CCTTG~AGGT 40 0 G~CTTCa.GTa C~GGCAGTCG QGAGCU~TC GSU4G1i:TGGCC TCAGCTCTTC TGGTGGCCTC 41qO

AG~C~ CC~TC~'r~ C~CC~ Ct:: ~CCTCCAG~ AGAAGAGCTA CaGGGAGTGA 4200 ~30 AT~:TGTCAC CIU~GAGCTTG TCTCTGGTCt: CAGATGTCA~ GGC~G~A TCCI~GTGCTC 421;0 AGAGCCCOG~ G5TCAGGCGC TT~TCCTC:CC TGG~CCCCAC CT~GCTCC:C TTCTTGGGAC 4320 36 q~GAGGAGBCT GTaTCA~ GCTCA5~ATTT CTGTCCCQ~ GG&TCCCCAC TGCTCATCTC 4380 ~TTATAGTC~ TCI::TGTGAGC T'rAC~TQCA ATTCACTCAC ATTTGGTGCT TCATGTTGl`A 4440 TT ~t;TTG C AGGCTCCTGC C~CCCTACCT CTBTCTCTGA G ~ CCTGT GA QGGGTGT 4500 ~ WO 93/22431 PCr/US93/03993 ' 2134670 ~1- .

TTCCG~G~CC TTC~TTTTG AAAT QT~GT C~GGGTCCTA CTC~AGTAAT GAGCAGC$CC 4560 CTGTG~TTT CTAAT¢GCCT GAGAAACCCC ATCTCTCAAC aTCATA~CCC TCCCTGTCAG 4620 T~AC~G~GAC TGCCCO~TCA CTGGT~CTGT GAT~TAAGTT TCTGCTCATG TGATGTCTTT 4680 GCTTTCCr~G A~CCTCTTGG CTTC~rTGTA ATTT~TAA~T AAAGCAÇGTT TATACATAAT 4740 AAAATTTTCC ACGTGGATTT TTTGT~GCAA TGT m TAAT ATAG~AATTC TGTGGCCTTG 4800 0 CTAGAGA~GG CATCATT~a GTTCGCTCTC CCAGGTC~AT AT5TCTT~T CTGTTAGTAT 4860 ATAGTTTAAA STTAAGTTC~ CATT~TAAAT TAATTTCAAT AACTTTTT~A ~TAAAATAGA 4920 ATTCCATCAR TTCCCCCCCC T~CATTTTTC ACCTGCCCAG ATGTCrTCAC TCCAAACCCT 4980 C~C~TGTTT~ TCCATTTT~A AA~T~AcAas C~TTTaAGGA AGCCTAT~TT TCCTTCATTT 5040 TCTT~TAa~5 AATTT$a~RA ~GTATCCAT5 TCCCTTTCTT TAAAGATAA~ CAAC~GATGT 5100 :20 cA¢TrcAGo¢ ~c~ccc~ cATaA~ ;c CTTCCTGTCA GCU~GAACAT GP~TCTGCl~G 5159 (2) IN~ORN~TION FOR S~Q ID ~O~:

(1) SXQU2NC~ CHARACT~RIST~CSs ~A) L~N~T~s 16 ~no ~cld~
(~) SY~Es u~i~o ac~d (C) S~R~NDYDN~SSs ~in~l~
~D) TO~OLoGYs l~n0ar (li) ~GL~CUL~ SYP~s popt~de YP0T~TICAL: NO

(iv~ ANTI-S~NS~s NO
~5 WO 93/22431 PCI/US93/03993 ~.~
213~167~ ~ ~

~xi) SEQUlSNCI!: DESC3?IPTIONs S~Q XD NOs4s 5::ys S~r Ser Val Ly~ Pha Val 5er Thr Thr Tyr S~r Gly Val Thr Arg :,~
::
:

.

.:

`: :
, :~ : ~ ::

~':
: .

.

Claims

-43- What we claim is:

1. A loricrin constitutive vector for efficient expression of a nucleic acid sequence in epidermal cells, comprising:
a 5' flanking region of the loricrin gene, said flanking region including a TATA box, a cap site and a first intron and intron/exon boundary all in appropriate sequential and positional relationship for expression of a nucleic acid cassette;
a 3' flanking sequence of the loricrin gene; and a linker having a unique restriction endonuclease site at the location of the start and stop codon, said linker connecting the 5' flanking region to the 3' flanking sequence and said linker further providing a position for inserting the nucleic acid cassette.

2. The loricrin constitutive vector of claim 1, wherein the 5' flanking region is approximately 1.5 kb, the intron is approximately 1.1 kb and the 3' flanking sequence is approximately 2.1 kb.

3. The loricrin constitutive vector of claim 1, wherein the unique restriction site is selected from the group consisting of Cla I, Not I, Xma I and Bgl ii, Pac I, Xho I, Nhe I and Sfi I.

4. The loricrin constitutive vector of claim 1, wherein the linker is a poly-linker, said poly-linker including a plurality of restriction endonuclease sites.

5. A keratin K6 inducible vector for regulated expression of a nucleic acid sequence in epidermal cells, comprising:
a 5' flanking region of the keratin K6 gene, said flanking region including a TATA box, a cap site, a first intron and intron/exon boundary sequence all in sequential and positional relationship for expression of a nucleic acid cassette;
a 3' flanking sequence of the keratin K6 gene; and a poly-linker having a plurality of restriction endonuclease sites, said poly-linker connecting the 5' flanking region to the 3' flanking sequence and further providing a position for insertion of the nucleic acid cassette.

6. The keratin K6 inducible vector of claim 5, wherein the 5' flanking region is approximately 8.0 kb and the intron and intron/exon boundary is approximately 0.56 kb and the 3' flanking sequence is approximately 1.2 kb.

7. The vector according to claims 1, 4 or 5, wherein the cassette includes a nucleic acid sequence coding for a protein or polypeptide selected from the group consisting of a hormone, a growth factor, an enzyme, a clotting factor, an apolipoprotein, a receptor, a drug and a tumor antigen.

8. The vector according to claims 4 or 5, wherein the plurality of restriction endonuclease sites are selected from the group consisting of Cla I, Not I, XMA I, Bgl II, Pac I, Xho I, Nhe I and Sfi I.

9. A method for in vivo transduction of epidermal cells with a loricrin constitutive vector comprising the step of contacting the vector with epidermal cells for sufficient time to transfect the epidermal cells.

10. A method for in vivo transduction of epidermal cells with a keratin K6 inducible vector comprising the step of contacting the vector with epidermal cells for sufficient time to transfect the epidermal cells.

11. A bioreactor comprising transformed epidermal cells including the loricrin constitutive vector of claim 1.

12. A bioreactor comprising transformed epidermal cells including the keratin K6 inducible vector of claim 5.

13. The bioreactor according to claims 11 or 12 wherein the loricrin constitutive vector includes a cassette having a nucleic acid sequence coding for a protein or polypeptide selected from the group consisting of a hormone, a growth factor, an enzyme, a drug, a tumor suppressor, a receptor, an apolipoprotein, a clotting factor a tumor antigen, a viral antigen, a bacterial antigen and a parasitic antigen.

14. The bioreactor of claim 13, wherein the nucleic acid sequence encodes proinsulin or insulin.

15. The bioreactor of claim 13, wherein the nucleic acid sequence encodes growth hormone.

16. The bioreactor of claim 13, wherein the nucleic acid sequence encodes insulin-like growth factor I, insulin-like growth factor II or insulin growth factor binding protein.

17. The bioreactor of claim 13, wherein the nucleic acid sequence encodes antihemophilic factor (Factor VIII), Christmas factor (Factor IX) or Factor VII.

18. The bioreactor of claim 13, wherein the nucleic acid sequence encodes an epidermal growth factor (TGF-.alpha.), a dermal growth factor (PDGF) or an angiogenesis factor.

19. The bioreactor of claim 13, wherein the nucleic acid sequence encodes Type IV collagen, laminin, nidogen, or type VII collagen.

20. The bioreactor of claim 13 for vaccine production, wherein the cassette includes a protein which induces an immunological response.

21. A method for ex vivo introduction of a loricrin constitutive vector into epidermal cells comprising the steps of co-transfecting the Hector with a selectable marker and selecting the transformed cells.

22. A method for ex vivo introduction of a keratin K6 inducible vector into epidermal cells comprising the steps of co-transfecting the vector with a selectable marker and selecting the transformed cells.

23. A loricrin gene of SEQ. ID. No. 1.

24. A loricrin constitutive vector having:
a 5' flanking region comprising nucleotides 1 to 1540 of SEQ.
ID. No. 1;

an intron and intron/exon boundary comprising nucleotides 1587 to 1679 of SEQ. ID. No. 1;
a 3' flanking region comprising nucleotides 4384 to 6530 of SEQ. ID. No. 1: and a linker to be inserted at the unique Cla I site at nucleotides 2700 to 2705 SEQ. ID. No. 2

25. A keratin K6 gene of SEQ. ID. No. 3.

26. A keratin K6 inducible vector having:
a 5' flanking region which extends from a unique 5' Xho I site up to nucleotide 360 of SEQ. ID. No. 3;
an intron and intron/exon boundary comprising nucleotides 928 to 1494 of SEQ. ID. No. 3.
a 3' flanking region which extends from nucleotide 4740 of SEQ. ID. No. 3 to a unique 3' Xho I site; and a poly-linker inserted between nucleotides 1504 to 1509 of SEQ.
ID. No. 3

27. A method for enhanced healing of a wound or surgical incision comprising the steps of in vivo transduction of epidermal cells with a loricrin constitutive vector, wherein said vector includes a nucleic acid cassette having nucleic acid sequence for a growth factor.

28. A method of enhanced healing of a wound or surgical incision comprising the step of in vivo transduction of epidermal cells with a keratin K6 inducible vector, wherein said vector includes a nucleic acid cassette having a nucleic acid sequence for a growth factor.

29. The method accodring to claims 27 or 28, wherein the epidermal cells are traduced with a plurality of vectors and wherein the cassette of at least one vector includes the nucleic acid sequence of epidermal growth factor (TGF-.alpha.), the cassette of at least one vector includes dermal growth factor (PDGF), the cassette of at least one vector includes the nucleic acid sequence for a matrix protein to anchor the epidermis to the dermis and the cassette of at least one vector includes the nucleic acid sequence for an angiogenesis factor.

30. The method of claim 29, wherein the sequence for the matrix protein is selected from sequences coding for a protein selected from the group consisting of Type IV collagen, laminin, nidogen and Type VII collagen.

31. The method of claim 29, wherein the angiogenesis factor is selected from the group consisting of acid fibroblast growth factor, basic fibroblast growth factor and angiogenin.

32. A method of treating skin ulcers comprising the steps of in vivo transduction of epidermal cells with a loricrin constitutive vector, wherein said vector includes a nucleic acid cassette having a nucleic acid sequence for a growth factor.

33. A method of treating skin ulcers comprising the steps of in vivo transduction of epidermal cells with a keratin K6 inducible vector, wherein said vectors include a nucleic acid cassette having a nucleic acid sequence for a growth factor.

34. The method according to claims 32 or 33, wherein the epidermal cells are transduced with a plurality of vectors and wherein the cassette of at least one vector includes the nucleic acid sequence of epidermal growth factor (TGF-.alpha.), the cassette of at least one vector includes dermal growth factor (PDGF), the cassette of at least one vector includes the nucleic acid sequence for a matrix protein to anchor the epidermis to the dermis and the cassette of at least one vector includes the nucleic acid sequence for an angiogenesis factor.

35. The method of claim 34, wherein the sequence for the matrix protein is selected from sequences coding for a protein selected from the group consisting of Type IV collagen, laminin, nidogen, and Type VII
collagen.

36. The method of claim 34, wherein the angiogenesis factor is selected from the group consisting of acid fibroblast growth factor, basic fibroblast growth factor and angiogenin.

37. A method of enhanced healing of a wound, surgical incision or skin ulcers in humans and animals comprising the steps of:
ex vivo tranduction of epidermal cells with a loricrin constitutive vector, wherein said vector includes a nucleic acid cassette a nucleic acid sequence for a growth factor; and transplanting said tranduced epidermal cells into the animal or human to be treated.

38. A method of enhanced healing of a wound, surgical incision or skin ulcers in humans and animals, comprising the steps of:
ex vivo transduction of epidermal cells with a keratin K6 inducible vector, wherein said vector includes a nucleic acid cassette having a nucleic acid sequence for a growth factor; and transplanting said transduced epidermal cells into the animal or human to be treated.

39. The method according to claims 37 or 38, wherein the epidermal cells are transduced with a plurality of vectors and wherein the cassette of at least one vector includes the nucleic acid sequence of epidermal growth factor (TGF-.alpha.), the cassette of at least one vector includes dermal growth factor (PDGF), the cassette of at least one vector includes the nucleic acid sequence for a matrix protein to anchor the epidermis to the dermis and the cassette of at least one vector includes the nucleic acid sequence for an angiogenesis factor.

40. The method of claim 39, wherein the sequence for the matrix protein is selected from sequences coding for a protein selected from the group consisting of Type IV collagen, laminin, nidogen and Type VII collagen.

41. The method of claim 39, wherein the angiogenesis factor is selected from the group consisting of acid fibroblast growth factor, basic fibroblast growth factor and angiogenin.

42. A method for treating psoriasis comprising the step of in vivo transduction of epidermal cells with a loricrin constitutive vector, wherein said vector includes a nucleic acid cassette having a nucleic acid sequence coding for a protein or polypeptide selected from the group consisting of TGF-.beta., a soluble form of cytokine receptor, and an antisense RNA.

43. A method for treating psoriasis comprising the step of in vivo tranduction of epidermal cells with a keratin K6 inducible vector, wherein said vector includes a nucleic acid cassette having a nucleic acid sequence coding for a protein or polypeptide selected from the group consisting of TGF-.beta., a soluble form of cytokine receptor, and an antisense RNA.

44. The method of claims 42 or 43 wherein the cassette contains the sequence for TGF-.beta..

45. The method of claims 42 or 43 wherein the cassette contains a soluble form a cytokine receptor selected from the group consisting of IL-1, IL-6 and IL-8.

46. The method of claims 42 or 43 wherein the cassette contains antisense RNA to TGF-.alpha., IL-1, IL-6, or IL-8.

47. A method of treating cancer of squamous epithelia comprising the step of in vivo tranduction of squamous epithelia cells with a loricin constitutive vector or a keratin K6 vector, said vector includes a nucleic acid cassette having a nucleic acid sequence coding for an antisense RNA.

48. The method of claim 47 wherein the squamous epithelia cells are selected from the group of cells consisting of epidermis, oral, esophageal, vaginal, tracheal and corneal epithelia.

49. The method of claim 47 for treating skin cancer wherein transduction of epidermal cells is with a loricrin constitutive vector and said nucleic acid cassette had a nucleic acid sequence coding for an antisense RNA
for the E6 or E7 gene of human papilloma virus.

50. The method of claim 47 for treating skin cancer wherein transduction of epidermal cells is with a loricrin constitutive vector and said nucleic acid cassette has a nucleic acid sequence coding for the normal p53 protein.

51. The method of claim 47 for treating skin cancer wherein transduction of epidermal cells is with a keratin K6 vector and said nucleic acid cassette has a nucleic acid sequence coding for an antisense RNA for the E6 or E7 gene of human papilloma virus.

52. The method of claim 47 for treating skin cancer wherein transduction of epidermal cells is with a keratin K6 vector and said nucleic acid cassette has a nucleic acid sequence coding for the normal p53 protein.

53. The vector according to claims 1, 4 or 5, further including a Vitamin D regulatory element.

54. The vector of claim 53, wherein the Vitamin D regulatory element is from the human K1 keratin gene.

55. A method for vaccination comprising the step of the in vivo transduction of epidermal cells with a loricrin constitutive vector or a keratin K6 inducible vector, wherein said vector includes a nucleic acid cassette having a nucleic acid sequence coding for a protein or polypeptide which induces an immunological response.

56. The method of claim 55, wherein the cassette includes a sequence for a viral capsid protein.

57. The method of claim 56, wherein the capsid protein is from the human papilloma virus.

58. A transgenic animal containing the vector of claims 1, 4 or 5 in its germ and somatic cells, wherein said vector was introduced into said animal or an ancestor of said animal at an embryonic stage and the nucleic acid cassette of said vector is only expressed in squamous epithelia.