Note: Descriptions are shown in the official language in which they were submitted.
.'~ 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 `~
-10-
.
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
-11-
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 .'~'`!,
-12-
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
-13-
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
2134670
-15-
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
2134670
-17-
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
-19-
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~ ~
-20-
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
-21-
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
IPLE 6
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
PLE 8
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$~: ~C-DOS~ DOS
5D) 80rrliARE) P~t-tltIn ~l~a-e ~I.û, V9~rl3t~0n i~
T APPLICiU~ON DA~A~
30 ~ (A) I~PP~ TIO~ R~
~ 8 ) F~LING DATlI~
(C~ CLA88Ir~ r~t
:: ~v1 ii) A~ Y/AG15NT INFO~ION:
: ~ 35 ~ A ) NA~: P~u l, Thom~ D .
; (B) ~aECISTRATION NU~BlSR: 32,714
~ ~ ~: (C) R~ NC35lDOCÆT ~ 2iUMB~SR~ D--5dOS
:~ :
,
WO 93/2243 1 PCl /US93/03993 .~
FZ 13.~G7 0 .
-2B-
( ix ) TEL~SC~M}~UNI~:ATION INEORMATION: '
(A) TEI~:PHON~t 713/651-5325
(B) Tl~L~FAX: 713/651-52~6
(C~ ISX- 762829
2 ) INFOR~ATION ~OR SEQ ID NO s 1:
~1 ) 8EQ~!:NCX CHARACTERISTICS s
(A) ~:N~;T~t 6530 ba3e pair~
~B) TYPEs nuclelc ncld
( C ) S~R~DEDNESS s double
(D) TOE'OLOGYs l~n~ar ~:
rp~: s D~A ( gsnom
( ~ll ) ~YPOI ~S~TC:AL s NO
: .
AN~ NSE 3 NO
:20
~ xl )~ 8BQU151~C:15 DESCRIPTION s SEQ ID ~O ~1:
25 GGATCC~ AT~G~GTCT ~GAGAC TATCCCGGGG C:AGC~AC AC~GAAGTGS~ 60
ATe~c~a TCA~;6GA~rG GGTGA~TCAC AGGGCCCCCA ATGT~AGC T~GAGA~GA 120
-:
ACCChGGG;~G = AT C:ACCTCAGTT CATACTGTCC AAP.CTGAAAC AAGTGGCAC)~ 180
AGTTTCTCA~: ~CA~Tt~ ATC:AGGP:r CX:TTTAGATC Al'TWaTGCTe CCCC~TAATT 240
P~:A~T CTGATT~G~h T2ATTCmC AACACJ~GCTG G~;q~GGAAC~A GGTTCI~ACAG 300
35 TGGTA'rCT7~A ATM~C:AACTG AGTTCCA~TG ATS;AAAt:AAA GGA~CAC TATGTTCTTC 360
ATAC:ACAGAG GGGGGGT~:T CTTGGCCCTA GGGTCATCA& AG~CTGAGT ~TCTTATA 420
GGAAAATAGT ~ GATGTCT TcAcAcAc~r c~rccAAT AGG&~C~AG GGC~GGCATG 480
~ . WO 93/22431 2 1 3 4 6 7 0 PCl/US93/03993
-27-
ATT~:AAGG~ AA~Gq~GTTCT GTCATGTGAG AA.AAGAGQA AAGTA~AAT AT~AC~TACT 540
ATGTAGTACI~ TTS:ATAmC I~TAAC~CCA Tl~C~ATGTT TCTGTGAA~T AAATTATAGG 600
5 ATTCC:TC;CTT GGTAG:A~CAA A~ATCA GACI~GCTCAA CAATGAACAA GTAGTC~GTA 660
ACTGCCC~GT TGGTGaC~TT GCA~AACTA CTGTGCTTTG CCCATG~;TGA CATAS;CTTGA 720
A~T~T~ATG GAAGACCTGA ACCCAACTaA GATCTC:TAAG TAS~TTCCAC TCTATGGTGG 780
0
CATCTC~aAG GTCAGAGTCA ~GTt;CAGCG CCATAGGACA TCAGI~ATCAA AGGGTCATGG 840
TGAAAAGGCT accAGGGTcT GTC7~GTTAG ~CTCACC~T TGTAAGTAAA GTI::AGTAGTC 900
15 AC;TI~ACI~AG ATCAAAACAC CTGCTCTQC AI~GGAAT~C TTAAAGl'AGA C~AAAGTt:AT 960
~CTAGTTACA GTGCTGrCST T~CCG~ A CCA~CCQAA CTGGG;F~GC$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$AGCT TC~.TGTC~T 1200
25 AGACAATGTe ~T~GATCq~CT AGATTCCCTA l~GGC~GCTTC A~:TCTGC AACCTAGTCC 1260
.
~CTI~G~ .G C~ C~G5~ aTG~cA~cA ACC~ ACAG AAGm~TGAi~ AACAATq~TCT 1329
.
GCCATCCAC~ CCA T~rCTAaTGA CCAAC~Gt:T CACTGl~ACA TU~GAGaAGT 1380
a:C,C~GTC~T ~C~C~A~T CCCTA~CCt:T ATCCCA~GAA l~;lUA~CT TCA~GAATGG 1440
GTC~ATCCTT CCCCTGCAAT CAC~ GG AGGT&CCTGA TU~aTAGAT~: AGTC~GAGCA lS00
35 ~;ACAAGAGT ATAAAACAC:A GGAGCACCAG TGTCCCTCAC AT~Gt:~TCA l:C:TCCTTCCC 156C
TC~CTCATCT TCCCT>CC l~C~ Tl~G TGTWC:CTCT CC~;GCTGTC TGGTCTCTCC 1620
A5TTGGCCT~ GCTCAGCTTG CAGAGAW~ AAS;GAACAGA GCCTTTCTCC CCTTTGG;A~G 1680
WO 93/22431 P~/US9~/03993
lii7~
-28-
GTACTCTGr~ CA~Ar~GAGA AGGGCT~TAG GAAAGCACTG GGAGAGTGGT ~AGC$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
0
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> ~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~
-30-
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
-31-
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
0
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
-32-
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
W 0 93/22431 213 4 6 7 0 PCT/US93/03993
-33-
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$GCAGCG CCA~AGGA~ TC~aAAT~ A~;GTCATGG f340
0
TG ~ GGCT GC QGGGTCT GTCTTG~G TTCTCACCTT ~GTAAGTA~ GTCAGTAGTC 990
: AGTAACAAAG ATGhAAACAC CTG~TCTCAC AAGGAATAAC rTAAAGTAGA CTAAAGTCAT 960
GCT~r5ACA GTGCTGTCT~ TTCCGTG~$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 ~>GCCTGA ~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
-35-
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)
0
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$CA GAGATAG:A(:C ATC;CQAGTA GC~CCTCI!~G TGACACAG'rA 4020
GTCT~ACCAC CACATTTTTA TCGATTAAAT GTA~ TAGCATGGTT ATATGTGCAT 4080
ATAATACACT CTGATTAC:TC ACTTC:CCTAT CCTTTCrrAC TCCTCCCCAT CCCAACCTGT 4140
W O 93/22431 . PCT/US93tO3~3~
21346~U ,.
-36-
AT~AATCCTT AC~TTCGCTA C~A~CCCTT ~ACCA~G~TT TTGTTAGTTT TGTTGGTTTG 4200
TTTTGTGACC CA~TGAGCTA ACQGGGCCA TCTGTATGAC CATGGGTTTG GATT~TGATG 4260
GA~TCCCACT GGG~ACACAA CTOAAACTAG T~ACTCCCCT TCAQGAATC TATCAGTAGA 4320
C~TAATT&A A~AGGGAATG GTGGG~CTCT CTCCATCC~T ~GCTAACTGT TGAC~GSACA 4380
a~cTTGTGcA GGCC~AaTGC ACACAACCA~ AGTT&CTGTG AGCTCATGTT TGCAAT~GCT 4440
GTGT~TaCA TAGGAOA~G TATTTTGGAG CCATTATCCA TGTCToGCTC TTATATTCCA 4500
CCT~CTCTTT T~GaATGTTC CTTGAGTCTT TGAGGAATGT IITGGTTA~A ACCGAGTGCT 4560
15 GACTTGTCAT rTATTTTCAG AaTCrTGAGC ATCAAA5GAT A~ATAA~ATA TTATATTATA 4620
W ~ TA A T5~TTG~A Q GA~ITTTCAT ATACC~TTC~ TATTGGr~A CCAT~ATCCC 4680
ChA5rSSSC~ CSCCTC~AAC AC~CCA~TGC TCCCA~ACQ G~T6AAACCT TTChACTC~ 4740
~0
TG~A~TI~CC C~C~IloC~T ~CA~r5TATC T~AITGTA~ C~CAACT CCCTT~ATCT 4800
ATCT ~CTAC CAA~CCCT ~TTCTAAACT GGTAGCCTAC ~ACTr~AGTT CCAGTACTTG 4860
ATGCAGAAGT A~ATÇGAGCA A~TGAAC~C ATGC$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
-37-
( 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$CA A~:CGCq~GG ~TCI~GQG~ GTGTCCG~GT GCCGCTCCCG 540
,
~;aGcA~;c~t;~ ~GC2CC~C;TI; CAI~Tt;~GTGG aGGAGCTGGC TTTGGCAGCA GGAGCCTC:TA 600
TGG:TGTG&a~ AÇ;CTCCa~CJ~ .T~CCAT CGG~G ~ C:AGCTGSeGCA TTGGAGGAGG 660
~TATGGCAGC C~ATTTGGAG CAA~CTTOGG CATTG5AWT GGAG~TGGT~ GTGGCTT~GG 720
35 CTT~GG~GGT G4A~C~G~CT T~G~TGGTGG CTAT&G&GGA GCTGGCTTCC CGGTGTGCCC 780
ACC~GGAGGC ATCCAAGA~G TC~CCA~CAA CC~GAGCCTC CT~A~CCCC TG~ACCTGCA 840
..
~AT~GACCCC ACCATC~AGC GGGTGAGGAC TGAGGAGAGG GAGC~GATCA AGACCCTCAA 900
W O 93/22431 . PCT/US93/03993 ~.
~134670
-38-
TAACAAG~TT GGCTCCrTCA TCGACA~GGT GAGACATGGT CCTCCCTAGA GCACCCTGTG 960
TGTCT~CAGG GAATGCTGAA ~AGAGGT&TA GGGAAGAGGC TTCAGTCTCA GCTCTG~TAC 1020
TGCCTGTGTT GCTAGTTGAT GCTCTGTCCT GGTTTGTGTT CCTCTT Q GT TAG~CTGGCA 1080
TCTGGAAATC AGGGTCAGCG TTCCTCTCCT CCAGAGGTTG CCCTATAAGG GTGTCTGGTC 1140
CCAGTGGACT GAeATEACTT AAAGACTCAC AAA~CAGGCT TGTAGGGA~ TGGAAGATTA 1200
T~AFTATGTA T~GTGCAGTT GGGAGGCA~G C QGCCTCAC TAAGCTGCAG CACACTTCAT 1260
CAAGCCATGG CTAACCTGCC AGTGCCCTAC ATGAGTTCTC TGCCCTCCTT AGAGAGGTGG 1320
15 CATTGGG~GC TTCAG~CTGG ACTGTr~CCC TCA~ACCGA& GGTCAGGGTC TAACTACACT 1380
: ~ :
~ GA~G~CS~T AGT~aG~CAG CC5~A5AG&G TACACACACT AG$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$CCAATA T~GTG Q CCT 1860
~TCCTCTTGT AGATATGAAC ~TGAAAT~AA CAAGCGCACA GC~GCAGAGA ~5G~AT~CGT 1920
G~CCCTGAAG AAGG~GAGTT GACTA~CCAC AAGGATGGGT TTCTCTG~GG ~A~GACATAA 1980
AAGGCCTTGT ATA~CTGCGT CATTCC~GAG AAAT>GGT TACAGGGAAA G~AGTGAACG 2040
GTCTGS&GAA GAGAGGTAAC CTGAr~CCAT GTTCTTGATG GTTTTCTCAG GATGTAGATG 2100
~ ~ WO 93/22431 2 I 3 g 6 7 0 PCr/US93/03993
-39-
CTY;CCTACAT GAACAAAG~ GAl~CTGCA~G CCAAGGQGA CAGl~CTAAQ GATGATATCA 2160
ACTTCTTGAG AGCTC~CTAT GAAGCAGTAA GCCCCCCT~G TCTT~CTTC TCC~TTCt:AT 2220
5 ~CACCACTCC CT~TA~TT TSCCCCC~GG GCAAAGTt;TT TGACCTCTC;C A&TTCT~A 2280
S;ACAAAGATG ACTATGGCTC TTTCTG'rCCT GC~G4AI~CTG TCTCAGATGC AAACTCACAT 2340
CTQGAQCA TCTGTGGTCC TCTCC~TGGA CAACAACCGT AGCCTG~;AC:C TGGACAGCAT 2400
0
CATCGCTGAG ~TCAAGGCCC AGTA~GAGGA CATTGCT QG AGAAGTCG&G CTGAAGCTGA 2460
~TCCTGGTAC CAGACTAAAG TaAGTATTGG GGTGG~GGCT GATGGGGATG CCTGGGGTCC 2520
ACCCTGAACT C~A~GAGTCT CTG~GTTCAG ~Ar5GGAGGC CCACT~A~AG AAAT~GGGAT 2580
.
~ ~ GTT~TCCGAG AhAAIGCACT GTGCACATGT ACC~TAGAAT AATGm~c TCGAAGAGTA 2640
: ~AAGA~CAo~ G~GG~AGAT& GAAAGITGCC ATAAATGG W TCCATGCTCT T~GCTTGAGC 2700
2~ :
aA l~ TGa~q~CC T~:AAA Çq~GAGAACA Tq~ TC CTG~GGG~ C 2760
TATGGAGTCT GTGG~ CC~ AAAoCT~TC T5GAGGAA~A GCCAG~AC~T CCATG~AAGT 2820
: 26 GT~GCCACT ~AGAG~GGG TTr~GT~ccG CATGT~ACAA CTC~CATAGA TaTCCTCTCT 2880
T~GATTGoC~ T~CAG~ATGA GGAGCTGCa~ OTCA~AGC~G GCAG~CA$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~ ~ ~
-42-
~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
:,~
::
:
.
.:
`: :
,
:~ : ~ ::
~':
: .
.
