CA2174040A1 - Gene therapy for myocardial ischemia - Google Patents

Abstract

The transgene-inserted replication-deficit adenoviral vector is effectively used in in vivo gene therapy for myocardial ischemia in a protective way, by a single intracoronary injection directly conducted deeply in the lumen of the coronary arteries in an amount sufficient for transfecting all cell types in the affected region, including cardiac myocytes.

Description

rl~ ~Utll~- N~ LlritK;~ ~l~ c~rJ r~c~ l u ~ UJC~C ~
~1 tlO40 UC041.001 DV1 PATENT
GENE Tl~IER~PY FOR IIIYOC~I~DUL I~CHEMIA

Rebted ~nDlic~tion The pr~ ~ent ~pplication is ~ dNision~l of Applicatdon No. 08/396,207 filed 5 Febru~ry ~8, 1~9~.
RACKGRDUND
Field of ff~e Invention:
The ~ ent in~cntion rclates ~o a recornbinant ndenovirus vector which ~s u#d u~ gene ~er~py for myocardi~l ischcmi~, a rn~tl,od for producing s-rne, ~nd 10 a ~lc~hGd of providin~ myocardi~l pr~tiGn during rev~scularization or non-revawulariz~tion procedures with ~e use of ~e vector. The vector ~fficien~y expresses a tran~gene In the myocatdium.
~ hi~ invention was m~de with Govem T~nt support under Grant Nos. R01 HL4~343 ~nd K1J, HL031~0, ~warded by Adminis~ation. The Govemment has t5 cortain ri~ht~ In this invention.

Bad~ground of the Art:
Myocardial kchemia occurs when the heart rnusc~ does not r~cci~ an adequate blood ~upply and is thus deprrved of ne~osary b~els o~ oxy~en and 20 nu~ient~. The most common cause of myocardial ischemia i8 athercsc'e v~is, causin~ bl~ses in the blood Yessels (coronary arteries) that provids blood flow to the heart m~scle. Present bee~,~nt modalibes include pharn~ 3ic therapies, coronary artery byps~s sur~ery ~nd percutaneous re~/a~cularization usin~ bchni~ues ~uch a5 balloon an~ioplasty. In the ~ttin~ of aCute coronary 25 ~clusion (usually seconda~ to in-situ th,~,"~osis of a coronary a~bry se~mentpreviously narrowed by atherosclerosis) treatment o~ acute myocardibl ischemia is oft~n ~chieved by u~in~ thrurnboly~c a~ents ("clot bu~ters~ to opon the occluded ~rteries. Stand~rd pha",:~!~ic therapy is predic~ted on stra~ies that involve either increa~in~ biood supply to ~e heart muscle or decreasin~ ~e 30 demand of the heart muscls t~r oxygen ~nd nutrients. ~,Gr~ed blood suppl~Y tothe myocardium is ~chieYed by a~ents ~uch as calcium channe~ blockers or ~ 12 ' 3~ l~ 4a FR ~ E I ~S . BERL I ?~FR~;,C~ Z~ U ~
217104~

nitro~lycerin c~;...pounds fflat i.c-~e ~o diarnebr of di~e~d ~nerie~ by c~uain~ r~ Gn o~e ~,~tl- rnu~de In the artenal walls. Decreas~d demand o~ heart mus~e for oxygen and nutrlent~ is accornpl~hed oi~or by agents ~t d~ ase ~e he., ~dynamic lo~d on the heut or tho~e ~t decrease the 5 cont~ctile rc,sponsr of the heart to a siven hc...~namic b~d. Sur~ l tre~nt of kch~n- c heart disease is based on ~e b~;~,3sa of di~ed arten~ ments with s~ate~ieally pb~ed bypass ~ra~ (usually saphenous win or intemal ry a~ fl¢). P~r~ubneou~ a~cular;~bGn i~ ba~ed on ~e u~e of t~ tP ~duce the n~Towin~ in di~-a~ad Gotonary alteries. All of ~e 10 ~bat~ies are b~sed on the c~ tion of ~chernk ~ es as ~e prunary l,caG,~nt evidence, and 811 have limit~tions ~ ~eir e~l~ctkrene~s in this regardAustralian P~bnt Publi~ion N~. 27~02~2, Co~ ponding toW0~3J06223, discloses adenovirus vectors for e~r~ision of desired gene~ in musde cells to treat muscular dy~uphy and ~romboses ~ugh ~e '~02 applicathn ~li~lo~es ~denovirus type 5, it d,stl~ses s~J6r vec~or conslructs which ~rc us~d for the t~eatrnentofmuscul~r~o~phy T~x~s H~n Ins~ Jwm~1artiGleZ1:10~11 (1594) discloses ff~e adv~nta~es of use of adenoviral vector~ in mediating emcient direct ~ene transfer for prcYantill~ rest~nosis. ~n par~icular, ~is article teachcs ~at ~e Ad5 virus transf~t~d into 293 oolls is an ~be"l~ly u~efu~
vector for ~ene l,_nsfer in coror-a~y arten~s. H~rz 18(4):2Z2 22~ (1993) di-sclos~s ~e advantages of use of replication-defident ~enovir~l v~ rs in dir~ ene therapy. Like the preceding article, this article teaches general advan~es in use of the Ad5 Yir~lS. AtT1~nt~n Joum~l of Medical Science 30 (2):12~36 (19~3) discloses the ad~anta~es of use of recornbinant adenoviral vectors in ~ene l,~"~r. This article ~eachB~ ~ener~l ~dv~.,tz~es in use of adenovlral voctor~, direc~ intraYa~cular in~ion, and bFGF ~ene for treating coronsry o~u~ ~n. HowcY~r, ~11 of the te~chin~s o~ abo~e ~ocuments are too general to address the in vrvo expression effi~ency of a oertain vector in my~c~rdial ~,r~:~.tion.
In particular, non~ of treabnent mod~Wes of the prior art addresses the issue of protectiion of tl~e myocardium a~ains~ .ai~b d4m~ge wh~n ~P~ 12 '96 16:4~ FR ROEEI~S,EERLI~E~&C~R5213 250 702g TO 160~5a2~27~ t~31 2179~
.ia does oocur. Prot~3ction of heart mu~e against ~et.c~b h~l~ been demonstrated in ffle ~ttin~ of iscl~mk prff~nditionino. This phen~,.,Lnon occurs when the he~rt b a~posed t~ brief period5 of ~schemic stres~ prbr to a probnged isch~hlic episode- Dur~ng ~e brief pe.i~ls of ischemb, production 5 of 5pecific ~ ~l~ted ~e~r~ i~ induced. These stress factors protect ~e my~ ~fdium ~g~ins~ ~ub~w~nt ~nd potentlally more hamlful, prolonged i~emic episodes. To da~, t~ b to ~nduce ~e~e same hct~rs by pharnnacolo~ic n~3ans ~ve b~Pen un8~cc .6F~I.

~RIEF DESC~IPTION OF THE FIGURES
~ IGURE 1 i~ ~ schema~c figure which ~ho~ re~eue r~ "bir,dtio"
construction o~ a trans~ene encodin~ adenovirus.

SUIUNEARY OF THE INVENTION
The present invention ha~ exploitecl a ~ene therapy approach to treat ~art di~as~. An obJec~v~ of the pre~nt invention i~ to pru~iJe a IllcUlo~
providing myocardial protection in which a stress related factor is produced in the myocardium and is present at the time of i~chemi~a 60 a~ to probct the myocardium again~t subse~l~snt1 potelltially more hannful, prolon~ed ischemic 20 episodes. ThiS objectiYe conCun~ protectiYe effects, rather ~an thePpelltic effects on myocsrdial ischemia.
Namely, one impo~ant aspect of the present invention is a method o~
providing myocardial protection, c4n)p~ing: delivering a replicabon~ri~ ent adonoviral vector to a myooar~lium ~y intracor~na~ inJection into the coronary 25 arteries, preferably ~ sin~le i~,je~tion of t~ ~ectorl di~y into the coronaryarteries, 50 as to transfect cardi~c myocy~es, which do not under~o rapid turnoYer, in ~e affected myocardlum, ~aid vector cornpri~ing a transgene Godin~ for ~ sbress re~ted hcto~ quch ~ hea~ sho~k prot~in8 HSP70i, ~SP~7, HSP40 and HSP~0, and the ad~nosin~ A3 r~oep~o" and expressin~ the 30 transgene in the myoc~rdium, ~ereby raisin~ ~e kvel of stre8s related factot in the a,,~ed r~ion of t~le myocardium. By injedin~ th~ Yector 8tock ~PR 12 '56 16:4S FR ROBB:~S,BE~L;~'ER3CRR5213 Z50 7~ IU 1~4~ 'Y~ '.W/~l ~ ~ 7~04U
conbinin~ no wild type virus deeply hlto th~ lumen of the ~~ronary arteries, pn~rably into bo~ the right und l~ coron~ry ar,terio~, of ~ myoc~rdium pr~ferably in an ischemic m~i~u~ prefer~bly in an ~mount of 10'~-10~ viral parffclesasdet~ .i..cdbyoptkalden~y(morep~forably10"~10'2vir~l S partcles). iit is pos~ible to locally trJnsfect most of the oell~, ~ei-lly c~rdiac myocytes, ~ich do not u~r~o rapid tL,-"~ver, in th-e ..rr_~d ~Tly~;z,rdium with~ enes for ~, str~s re,'at~d hctor, Ul~r~')y m.~d",ki,.~ my~,rdial protec~ effic~:y of gene transhr, anc, minir"ki-,~ th- posslbility of an inflammatory r~pons~ to v~l prot~ins. If a v~r~t~icult,r myoc~te ~,~cihc 10 promoter is used, ~e vr~, "ot~r more securely cna~l~s expression lin~ited to the cardiac myocytes so as to er'e,~L~ avoid the pot~ lly ha,rm-ful effects of ~ngiogenesi~ in non~rd,ac 'd~su~s such as the retina.
In the above ~ th~d, myoc~rdial pr~l6cti~n i~ expecbd to be more effe~tive in cas~s that (a) said pati~nt ha~ no~r~ascularized ischemic heart 15 di~4~&e and said protection is de~ired durir~ pbnned non~.~iac surgery, who~in s~id veetor i8 admi"i~t~red a plurality of days prior to the pl~.",~J
n~n-cardiac ~ur~ery; ~b) said ~ ~io" is desired in a,lti~ ion o~ complex perwtaneous revaseulari~ation, and wherein sai~ veetor is delivered at the time of a dia~nostiie cathebriza~ion a plura~ity of days prior to the revaseulali~ation;
(e) said pro~eetion is desire~ in ar,ti~p~tion of ~I~ ex cardiae surgery, and wherein said vector is d~livered at the time of 3 diagno~tie cardiae eatl ,et~riL~lion; (d) said prot~lio~ deail ~d in a donor heart to be transplanted into a host pat~ent w;th a coronary t~ 3, and wherein said vector is d~livered ~t the tirne of a dia~nostic coronary a~ , aphy prior to expbnation to rule out 25 coronary disease; and (e) said proteation i5 desired in a patient wi~h drffuse, nonrevas~ul~rizable coronaly attery disea~e, at ~e ~rne of ~ diagno~tie eoronary anaiography prior to explanation to rule O~n coronuy dista~, wherein said vector is d~liver~d ~ plurality of times.
Another aspeet of the pr~ent inv_nlic,l, is an ir~ ot: le adenoviral vector 30 preparation, cc~",~,.isi"~ a r~n~binant aden~iral vectot, p~f~ b~ in afinal viral titer of 10'0-10'2 viral particles, said ~edor containin~ no wild-type virus 12 '~6 16:4~ -~ ~B~ 3t~ t~r~l~c~ U lb~ C(4 1~

and compri8in~ a putial ~donoviral sequenoe thm which the E1AIE1B ~enes have been .Iclehd, ~nd a ba~ne codin~ for a ~tress rel~bd faGtor ~uch as he~t 8hock pr~teins HSP70i, tlSP27, HSP40 ~nd HSP~0, Dnd ~e adenosine A3 ,~pt~r, d~en by a p,~-.ote~ ~nked by ~e panial adeno~irat sequenoe;
5 ancl ~ ph~rmaceutical~ ~yt~ble carr;ier. By u8in~ tlli8 Ini~ctable adenoYiral v~ctor preparaffon it i6 ~os6ib!e to p~ff~", effec~fe ~denoYirus-mediated stres~ rela~ed factor-codin~ ~ene tran~fer for the t~e~r~ of dinical myoe~rdlal i~mia wi~out any undesirabb ~ects.
A furt~or ~pect of ~e present h~nffon i~ 9 method of producbon of a viral stock oontainin4 a rcct;.,l~i,.ant ~ector capable ~f ~ r~sing ~t~ b7 ~o in th~ myocardium c~-,~.~ing the st~p~ o~ clonin~ a *s " ~ "; ,~
trans~ene coding for 9 stress related factor such a$ heat ~hock proteins HSP70i, HSP27, HSP40 and HSP60, and ~e ad~nosine A3 re~ptor into a plasmid containing a promoter snd a polylinker flank~d by partial adenovial 15 ~equ~nces of the left end of the human adenovirus 5 ~enome from which the E1ArE1B ~enes have been d~leted; co tra"sf, ctir~ said plasrnid into mammalian oells tran~fuin~ w~h the E1A/E1B genes with a plasmid which contains the entire human adenoviral 5 genom~ snd an additional insert makin~
the pla~mid too br4e to ~e enr~psid~ted whereby re~cue r~c~mbin~tion takes 20 plac~ beh~een the trans~ene-inserted plasmid and the plasmid havin~ the entire ac~enoviral ~en~me so as to create a recombinant ger,o-.,e containing thetrans~ene without the E1AJE1B genes said r~co,~binant genomQ befn~
suffioiently smali to be en&~psid-ted; identifying su~essful recombinants in cell cultures; propa~atin~ ~erewlting recombinant inmammalian cells~ar,~...~d 2~ ~ith the E1AIE1B gen~ nd purifyin~ th~ propa~ated recon~i..ants so as to contain tte recombinant ve~tor w~thout w~ld type virus therein.
Based on the pr~nt invention ef~ ve proteolion to the hearS muscle against myocardial isch~rnia su~ as th~t encountered durin~ threatened myocardial inf~r~tion ~h~art ~ttac~c) can be surprisingly achieYed. That is the 30 present inve.l~ion ~llow~ for pro~ection ~gainst ti~sue n~uosis ~econdary to prolon~ed iscl~ k ~ des. Technica~l detail~ are deline~te~ ~elow.

i~FR 12 ' 56 16: 5~ F~ ku~ t~ir~ H~cl~ c~ c~ l u lW~Cr~CYJC ~ JL
2:17~0~1J
DETAILED D~SCRI~TION OF TliE Pf~l ~ tRRED EM~ODiMF~TS
Tran~g~n~ Codin~ for Str~ lat~ Factor In the pre~ent i~ tiC n, vnrio~ str~a~ rel~d hctors wl~ich are capable d prot~in~ ial i~chemia can be u~d; heat shock prGtcins HSP70i, HSP27, 1 lSP40 and HSP~ nd ~ ntkns ~ine A3 receptor can be ax~ l~d. Adc~,osi"c pl~ n ir.,por~nl role in mediatin~ ~e ph~no",enon of ischemic pr~co~itioning. the funcbon of adeno~ine appears to b~
me4bted v~a A3 typ- adenosine ~c~t~. In cell culture ~xpe, i..~n6 in which ~e numb~r of A3 ~Up~or~ per oell w~s inw~ed, the efficacy of an ~der~ine anolo~ue (Gensia Phannaoeuff~l) to miti~ate protecton a~ainst i~chemia was increa~ed. Th~ codin~ r~ l5 for ~e~s hctors are known in the ~rt, and it is possibl~ to download these cDN~ s~quences from G~nebank and other ~t~h~nks oY~r tl~e int~ , for examp~. ~ull or partial len~th cDNAs coding ~or the aboY~ factors can be usBd in the present inv~ntion. Other than 15 a~ove, sarcoplasmic reticular calcium AT~ase can b~ used f~r the purpose of studying myoc~tdial calcium handling/hy~ phy.

Help~t Independent R~plication Deficiont Human Adeno~irus 5 Sy~tem The cDNA o~ t~r~,~t isbrarisf~.,d,d tothe myocardium, includin~ cardiac 20 myocytes, in vivo and dire~ cDnstitutivo production of the re~ncoded protein. V~ral vc_t~ provide ~ mean~ f~r highly emcient ~ene ~nsfer. Sever~l dfflerent gene transfer approaches are h~ ble. The present inventor~ initially used ~e hslper-independent r~pl,c~tion def~ient human adeno~irus 5 system which ha~ preYiously dc. ,-on~t~a~ t~ns~ion ~reater than 60% of myocardial 25 oells u~ vr~ by a ~ ie intracoron~,~ in,ec~on. Non-repli~at~ve recG..,binant adenoviral ~cto-~ are partiwlarly useful in l~&ns~cti--~ wronary endotheli~m and cardiac myocytes resultin~ in hi~hly rfil,ic"~ l~an~r~ction aner intrsvenousiniectbn. The ~om~i"ant adenoviral ~ectors based on the human aden~virus S (KjrDlo~y 163.614~17. tg88) are ",i~sin~ o~a~l~dl early genes from the 30 adenoviral ~e.~r~ (usually E1AIE1B). and are ~ere~ore unable to replicate unkss ~rown in "permissive" c~ll lines that provide the mi~sin~ ~ene products ~PR 12 '9b 1b:50 FR ~0~EIl~S,~LlNtl~ icl~ J~ acy IU lb~l4b~c~c~1 r~
217~0~0 in trans. In place of th~ mi~in~ adenoYir~l ~nomic sequcn~s, a bansgene o~ int~rest can be cloncd and will be ~xprossed in tissueloells in~e~d w th the replic~on defident adcnov;~ ~Js. Aithough adeno~rirus~ased ~ne tld.-sf~r does not result tn inte~ration of ~o b~c"cnc into U~e host ~eJ)GI~le (less than 0.1%
5 adenovirus .--ed,atl~ tran~ on~ re6ult in trans~ene Tncorpo~a~G,- into host DNA), and U .erc.fo, ~ i~ not stabb, adeno~firal v_ct~r~ can be pr~pa~bd to hi~hti~er and allow gen~ tran4~er to no~r~plTcating cell~. Althou~ ~e t,ans~ne T~ not l ~s~se~ to daughter oells, Tn ~he ca~ o~ the aduK c~rdiac rnyocytes, which do not divde, this is not ~ opoi~nt limitafion. Retroviral v~ r~
10 provide stabb ~ene ttander, and hT~h ffters ~re now obtainable via retrov~rus ps~udot~ing (Burm, et al., Phc Natl AC~d SGj (USA~ 8~:8033-8037, 1~3), but current r~ov;.al vectors ~re unable to transduce nonrepli~t;"~ oells (adult cardiac myocytes) emcbntly. In addition, the potential hazards of transgene ;"cc,~GI~tion into host DNA are not warrantsd if short~terrn ~en~ ttansfer i~
15 u~cicnt. Thus, a limited dura~ion expression of a stres~ related factor may be ~uffic~ent ~or bmporary my~cardial prot~lion, and transient gene transfer for some c~rdiovascular r~i~e~se pro~ses may be adequate and possibly prefe~able.
Human 2~3 oells, which ar~ hum~n embryonic kidney oells tra"sfo,-,-ed 20 wffl adeno~irus E1~JE1B ~enesl typil~ the permissive oell lines. Howe~re~, other oell Eines which aUow r~plication~defic~ient adenoYiral veators to pr~pa~ate erein can be used. Thus, other oell lines u~eful for this purpose include HeLa oells.

25 Con~trucbon o- Recombinant Adenovlr~l Y~cto~
~ 1 adenoY;iral vectors used in the present inv~r,t~n can be constructed by ~h~ r~scue r~co nbination te~hnique developed by Fr~nk Graham (Virology 163:614 617, 1~88). Briefly, the ~an~ene of int~r~8t ib clon~d into ~ 8huttle v~tor that contains a promo~er, polyl;nker and pa~ial nanking adeno~iral 30 sequerce~ from which E1AfE1B 9enes }~ave been deleted. AS the ~hunle v~tor, ~la;.",i.~ pAC1 ( V~rolo~y 163:614~17, 1~88) (or ~n analo~) which ~PR 12 '96 16:50 FR ROBBI~,BE~LINER~C~RS213 25~ 7EZq TC 16~4682~Z,'~
21740~0 encodes p~ ~ona of ~e ~t end o~ the human ~d~novirus 5 ~cnomc ( vir~logy 163:~1~617~ 1~88) minus th~ early pr~;., ~.~oclin~ EtA and E1 B sequences ff~at u~ es~ential for virsl r~pl~tion, and pl~smid ACCMVPLPA ~J 8iol Chem 2~7 :2512~-2613~ 2) which conbins polylinker, ~e CM\I ~r~Gtu ~nd SV40 polyadenyl~on signal tbnked by putial adenovTral sequ~nces from which the E1AJE1B ~en~ h~ve boen debted can be exemplified. The u-~e of plasmid pAC1 or ACCMV~lA facilibtes ~ cloning pro~ss. The shuttle wctor is then co transf~ h a plasmid wîlich contains the entire human adenovir~l 5 genome wffl ~ bn~th too br~o to be en~p~ ted, into 2~3 cells.
C~ ~tior, can be cc~ndu~d by cakium pho~phat~ precip~ion or ipor6ctiGn under cGndfflon~ such as tho~e ~.so~d by Bio~e~h.,iques 1S:8O8-872, 19g3. A~ ~e pla~mid havin~ the entire adenoviral 5 ~enome, ptasmid JM17 whic:h encodes t~ en'dr~ hu7nan adenovlrus 5 genome plus portions of the vector pBR32~ including the ~ene for a~pToillin re~istanc~ (4.3 kb) ~ cxe.~ though JM17 encodes ~ll ~ the adenovi~1 pr~eins necessary to make mature viral particles, it is too large to be encap~ ted (40 kb versus 36 kb for wild type). In a small ~ubset of co transf~ d oells, rsscue r~combination ~t~ee., the trans~ene ~G"lail,ing the shuttle vector such as plssmi~ pAC1 and the pbsmid havin~ the entire adenoviral 5 genome such as plasmid pJM17 takes pl~ ~o ~s to ~eate a recol"l.~,lant genome that is deficient in the E1A/E1B s~c uenoes, and that contains the bran~gene of interestbut seeondarily loses ~e addKional ~equenoe ~uch as the pBR322 s~quenoes durin~ rscom~i"~tion, thereby being small enou~h to ~e ~ne~r~ t~ ee fi~ur~ 1). Wlth r6spect to Ule above .-~tl~d, we haYe r~po,~d sl~c~sful results (Giordano, et al. C~ 1/8~'10r7 SJ:1-13~ 93, and Giordano and lI.~.,n~ond, C/in Res ~2:123A, 1994). The CMV driven ~alacto~ida~e ~ncoding adenovirus HCMVSP1LacZ (CLINRES (Ab8) ~2:123A, 1~94) c~n be us~d to ovaluate effi~r~y of gene tr~nsfer u~in~ X~al t~_~t~e"t.
The inKial mode of gene transfer uses adenovir~l vc~t~r6 as dEl:neat~d ~bove. The advanta~es of these ve~tors inctude the ability to effect high efficiency ~ene ~nsfer (more ~an 60% of t~rget or~an cells transfe~ted /n ~ 12 '3~ 16:5~ FR R~B1~5,Btl~ t~a~H~,cl~ u l~lU4t~ 4 217~0~
~7vo), th~ e~e of obt~inin~ hbh t~or ~ l St~5 and ~e abili'ty of t~ v~c~ Jr~
to eflect gene band~r into colls ~uch ~ cardi~c myo~ which do not rgo r~pid tllrno~w. One ~ot~l di8~dvant~o~ i8 that the cunsnt ~ener~tion of this vectol does not result in ~table gene tr~n8fer. Genes 5 tran~ferr~d to the rnyocardiurn b~y adonovin~ veclors do not htegr~b into the host oell DNA and, ll~r ~fur-:, do not ~t ~e~ on to ~e pro~ny of dividin~
oells ~ribr~blasts, cnd~aal ~Ib, 8~ JUI muscb cell8, etc.). Genes transfe,,.~ to~emyocardium bycurrent~.~.~tiGn adenoviral ~f~ct~lra remain ~ctive c~nly for a period of weeks to ~.IGI~U.S. This may actually be 10 advanta~eou~ for cerbin clinical awlications swh as myocardial pratection to induoe a contro~led amour~ of a ~tress related hctor.
Alterr~ti~l~f, newer ge.,~ on adenoviral vectors that have further dcl~o,.~ in ffle adenoviru~ ~C.~GIlIC (in ~ddi~io" to E1AIE1B) are under development. These v~ctor6 have the pot~ l to effect longer term gene 15 tran~er and to be l~;~ immuno~enic. tf iit i~S d~t~n-li-l~ that longer b~n ~ene transfer would be more emc~ci~l~s andlor innan,~ ory response to fir~t generation vectors becomes probloma~c, these newer generalioi, vectors could be used. In addition, if gene transfel limited tD ~e ar~erial w~ll prov~s ~s efficaoious as myocardial ~ene transfer to effect myocardial p, ot~ ;tion, 20 alternative method of gsne transfer could be u~ed includin~ ctropor~tion, useof hydro~el coated balhon catheters, use of li~osor"~s or use of altemate viral vectots includin~ retrovirus or adeno ~soc;~leJ viral vectors.

Card~c-Speclflc Promote~
~5 It is also propose~ in the pr~3nt Inve. tio-~ to use oell tar~eting not on1y by ~ .y of b'le b~n~gene into ~e coror~ry utery, but a~so, in add~tional exper~nls, by usin~ a ventri~ular myocyt~-speci~c pro~not~r. By fusin~ the tissue-speciflc ~dns~ripli~nal con~ol sequences of leR v_ntricular myosin li~ht chain-2 (~JILC2V) to a transgene such as the FG~5 ~ene ~1vfflin th8 adenoviral 30 construct, ~ans~cne ~r~ n i8 ~imited to ventriculsr eardiac myocytes. The effic~cy of ~ene oxpr.~sien and degree of ~ if~ity provided by the MLCzv F;PR lZ '96 16:51 FR ROBBII';S,BE~LINER&CRR~213 Z5a 7a~9 TO 1~4t~M~4 i'.l~
217~0~
promoter with lacZ h~ve bean det~l",;ncl, u8ing th~ r~n~bin~.~t adonoviral ~y~em of the pre~ent i~ a.~n. C~rdbc~peciflc expr~ssion hp~ been documc.~tcd previ~usly by Lee, t al. (J B~ol Ct~em 267:15875~158BS,18~2).
The MLC~V prorn~r i8 c~"-pn~ed o~ 250 bp, ~nd ea~ily ~ wi~in the 5 ~denovliral-~ p~hgin~ trsin~. ~hemy~sin heavy chain promoter, kno~n to be a vigorous promoter of tran~iption, eannot be used becsu~ h Iarge ~ee (~.5 kb) cannot m within ~e ~deno~ral vet;tor. Other p,ur"~t~r~, such as the tr~pu"..~C pranoter, wl~ile hi~hly ~fficacbus ~nd suffici~ntly small, l~cks -deqlJ~te tissue sp~ city. By usir~ the MLC~V prc n~otEr ~nd delNerin~ the trsnsgene in ~o, X i~ beli~ that ~e cardiac myoc~te alone ~that is wi~out cGncon~itant ex~r~s~io" in endoU,e~al c~ "~- muscle oells, an~
fibrobln~t~ within the heart) will provide ~d~u~te expression of a ~tress relate~
factor such ~s heat shock proteins HSP70i, HSP27, HSP40 and HSPB0, and the ade, .o~i"~ A3 .e~p~or to pron~o~ myocardial prot~ction. Limiting e~"ression to the c~rdiac myocyte also has advantages re~arding the u~ility of ~ene transfer for the treatment of clinical my~c~.dial ischQ~ a~ Ely limffln~
expression to the heart, one a~oids any pot~ntial~y harmful effect in non~rdiac ti~sues. In acJ~ition, of the oells in the heart, ~e rnyocyte would likely provide the Ic~ngest trar~ene expression ~inoe t~e oells do not under~o rapid tumover;
exprossion would not therefore be deusased by celi dNision and d~ath as would occur witt~ endotl-~ celk. Subsequent studies will determine whether tar~etin~ ~ene ~ r~ ion to the ~nd~U.elial oells, and limitin~ ~xpre~sion somewhat to the coronary endothelium by intraoorona~ injec~ion, will be a cient means todeliver thetransgene. Endothelial~ fic pror~ot~r~ are already availabb for this purpo4e (Lo~, et ~I., J Biol Chem 265:10446 1045~, 1990). AS yet t~e~e are no fibroblast or ~ ootll mu~cle oell promoters avaibble that would effiebnt~y limit expr~ion o~ the t~ ene to ~mooth musde or hbr~ ts w~thin the he~rt.
in the present inv~ntbn, tugeting ttle he~rt by int~ runary injection with ~ high ffler of ~e vec~ nd ~a"~r~ g ~ll celi types can maximke the probability for succes~. Namely, it is ' elie~d th~t a more d~ tic result can -1~

PPR 12 '9'o 16:51 FR R~EI~5,~LI~Ll~ ~ IU lO~ r~lc~l 21740~0 be ~ch~ev~d if not only myoc~t~s but also cell types other ~an myocytes ~r~
eted ~s ~vell, aW~ough th~y are divid~ng c~

Prop~-tion and Purificatlon ~f AdenoYi~l ~ ctor S ~l~c3hl r com~inant va~t~ can be plaque purified acoording to ~andard l,.stl,ods. The re~ulting viral va~,~. ue prop~t~ on 293 cells which provide E1A and E1B fi."ution~ in tr~n~ to ~r~ in the 101~-10'7 viral part;des/ml ran~c. Cells can be infected at 80% c~ntluenoe and harve~ted 3t 36 48 ho~lrs post~nf~ n. Aner 3 freez~aw cycle~ the celtul~r dobris is pctleto~ by ~ndard oentr~ation and ~e vinus ~urther purified by C~CI
~radient ultr~oentrifu~ation (doubb C6CI ~radh~t ultracentrifugation is prefened). Prior to in ~ injection, the ~iral docks Jre ~o~ by gel filt~iGn throu~h sepharo~e columns such as G25 sepha~ex. The resultin0 viral stock has a final viral titer in t~e range o~ 10l~-1012 vi~al particbs/ml. Th~
adenoviral construct must be hi~hly purified, with no wild type (pot~ntially replicative) virus. Impure constr~cts can cau~e an intense immune response in the host animal. From this point of vi~w, propa~a~on ~nd puri~cation must be conducted to exclude contaminants and wild-type ~nrus by, for example, identifying succ~ssrul recombinants with PCR usin~ appropriate pri~ners, conductina two rounds of plaque pur;fica~,l, and double CsCI ~radient ultracentrifugation.

D~l;very of Ri~c~.nLin~nt Ad~noYir~l Vector~
Th~ vilra~ stock can be in the ~orrn of an injec~ble ~.r~pa~tion o~ntainin~
pharmaoeutically ~orA~pt-~b ~arrier 8uch as ~alin~, a8 I~;O~ Ary. The flnal tibr of the voctor in ~e injectable preparation k prebrably in the ran~e of 10~~-1 o~2 viral particb8 which allows for ef~ ve ~ene transfer. The adenovirus tr~n~Dene constructs are del~vered to th~ mro~rdium by direct intra~oronary in,~tion u~ing shndard pereubneous c~tl ,~tcr based methods under fluorosc~pic ~uidanoe, at an arnount wfficbnt hr the trans~ene to be expressed to a d~gree which allows for highly eff~tive ~erapy. Th~e amount hrK lc ~b lb~ K KU~i~liN~t~Lil~t~~ ~Cl~ C~ C~ IU lwL~oac~c~ r~
217gO4~
of th~ v~ctor to be inj~ted i~ pre~r~bly in the r~nge of 101~-10" vir~ r~es (more pref~rsbly 10"-10 ~ vir~l p~rtick~). The injection ~ho~lld be m~de d~ply ~such as 1 cm within tho arterial lurn~n) irto th~ lumen of the coronary srterie~, and profer~bly be m~do in boUl ooronary art~ri~s, as ff~ growth of S collateral blood ve~s~l~ h hi~hly Yari~bb within indhridual patient~. By inJectin~
the n~dt~r~al diroc~y into the lumen ~f ~e coronary artery by coronary catl~l~,r-~, i-t is possible to target ~hB gene rat~er e~fec~ely, and to minimize loss of ther oombinant v~ to the pr~.in-~l aorb during i~ on. Gene expr~ion when deliver~d in this manner i~ rninirnal in ~ INer, ~nd vir~l RNA c~nnot be 10 found in the urin~ at ~ny time ~tter intracoronary in ection. hly variety of corona~y ~th~ter, or a St~ck per~usion ca~l,ettr, and ~o forth can b~ u~ed in the present inv~u~io,-.

Prohctiv~ Applleation~
The replic~tion deficient r~ombinant adenoviral ~veclors of the pre~ent invention allow for highly enident ~en~ ~ansfer in vn~o w;thout evidenoe for cytopathic effect or inflammati~n in the areas of ~ene delivery. 8ased on ~se result8, ~ ~ b-l eveJ that a high enough de~r~s of in vivo gene ba~fer to effect In vivo funct onal ch~nges is achieved. In particulsr, protective use of the 20 vectors can be advantageous. In order to provide optimal prot~lio" to the my~cardium, stress proteins must be pr~s~nt at the time of ischemia. This requires ~ene t~ansf~r prbr to ~.ltic4,ate~1 isch~"ia. Alfflou~h the timin~ o~
many pro~onged i~chemic ~pisrJes Is unpr~ic~ble, t~re are specific settings during which ;_d,c."ia ~s antiapated. These ci~cumstances specifical~y allow 25 for gene lr~ f_r prior to the ischemic event. ~he followin~ includ~ some of the clin;c~l a~tLn~s in which ~ b for a ther~peutic ~ene ~ral~srer approach i~
~nl,ci~,~ttd:
1. Gene t~ansfer to proYide myocardial protection durin~ non~rdiac sur~ery in pat;~nts w~th non re~/ascubrized ischemic he~rt disease. This is a 30 coi""~on clinical problem which o~en requires a coronary rev~s~u~ ~on procedure (e.g., angiopiasty or bypa~ sur~ery) befors proc~Edi"~ wffl thé

I~KK lC ~b lt- ~ KK KU~5iN~, ~tKLlr~K~I_HK~cl~ C~U '~C, I U lW~IO~c~C f'l ~. 1~- ~L

non-carahc ~r~ery (hip repbc~nent, 9911 bladder ~ur~ery, dc.). If ro~ ular~za~on i~ not pos~ible bec~ the coronaly vasculature is dfflus~ly dise~sed or ~ rislt o~ c~rdiac sur~ry b thought to ~e una~ceptably high, tile non~rdibc ~urge~ en precluded ~us ~ in~ p~ffent to further 5 morbidity. In ~cse s ~n~s, ~en~ ~ans~er could be el~d by intra-coronary i~njec~on of the vkal construct se~ra~ days prior to U e pbnned non-cardiac su~ry 8uch th~t kvel~ o~ prot~ tre88 factors in the myocardiurn would be hi~h during ~e an~ated ~ur~ery. Cardiac c~U.~.~Uon, ns~ess~y for ~ene dcln~ry, does not require anesth~b and is very well tolerated by 10 othe~rise clinically oon~pr~ d p~lknts.
2. Gone t.al-~fer to provide rnyocardi~l ~,rut~ n durin~ complex percutaneous revasculareabon pr~ures (anghplasty. athe,~n~y, etc.) dufing which prolonged i~cl~.l,ia is ~n'd~ipabd. Percut~neous rev~scul~rkation of the coronary vasa~ re is compli~l~d 4% of the time by 15 abrupt to~l closure o~ the tar~eS vss~el. Alfflou~h ischemia can often be a~orted by u~e of i. ,t,~oor~ary ~rombol~kic a~ents, plac~.r~. ,t of intracoronary stents or emer~ent ~ypas~ surgenes, frequently ~s~ci~ted w;th irre-~cr~
myocardial dama~e. Even when abrupt v~eel cbsur~ does not occur, a si~nificant number of procedures are corr pli~ted by "810w-flow" ~econda~ to 20 non-ocelusr~e in situ lhron)bo~i~ or micro embolizatlon to the distal coronary vascubture (common when treatin~ dis~ased bypass gra~). These palie,ll~
are also at high ri~ik of peri-prooedur~l myocardial damag~. Usually, these patients undergo a ~iagnostic cardiac calhat~,i~tion several days prior to percutaneous r~vasullarization. Intracoronary gene delivery to the myo~rdium 25 at risk at the tim~ of ~e diagnostic c~U~r~lion m ~ntkipation of rev~s~?rizaticn in high ni~k p~tie.~t~ is beli~ed to be cons~ cuously effective. 3. Gene transfer ~ provide myoc~rdial p,~ n durin~ complex cardiac surgery (complex revascub, ~lion ptooedures, ~ralv~ surgery, wmplex congenital hsat1 con~ctive au~e, ~1 etc.). Coronary a tery bypa~s surge~ is 30 assoc-i-ter~ with a 3~.5% hc;den~ of peri~p~.d~i~re myocardial inhrction.
When peri~,~r~tiYe infarGtion does occur, peri~operative mort~l ty i8 hi~her and ~I'K 1~ ~b lb; ~ ~K KU~ , ~t~, . r~tK~ur~K~~ c~ I U lW~3c ~4 r . ~

in patients with r~sidual bl~ wntncular ~nc~on and tncompl~te wulariz~tion the bng-brrn pr~ is is poorer. Gene b~nsfer by in~coronary injectlon at ~e ffme of dia~no~Uc cc.Jizc c~ at on iust pnor to surgery 1~ believed to ~ pecially ~ve. We a"tbi~,alcJ ~is ~pproach 6 would be holpful bo~ in hi~h~r~sk v~e s~ ery and oci,~6nital heart di~a~e ~ur~eries.
4. Gene trangfer to ~de nnyoc~rdial prchc~io" to donor hea~
prior to cs~ c ~ansplanbffon. Damage to donor heart~ as ~ result of unav~idable dehy~ between ~e ame of explanation and the ffme of gr.~
10into the host patbnt i8 re~ponsible far a si~nific~nt proportion of ~nsplant rel~ted rnor~idity and hiled transpl&.~blion pr~l~res. Donor hearts o~en undergo dia~no~tic c~runsry nngiography pri~r to ex~ nat;o" in order to rule out coronary d;~,aYe. G~ne lla-lsfsr to ~ rnyocardium by intracorona~
i~lion at thi6 tirne is belie~ed to be partcu~rly eflective.
155. Gene bransfer to pro~nde myoc~rdial prot~ction to patients with dffluse, nonrev~s~'~zable coronary art~ry disease. A ~ubse~ of pa~e. ,~ with coronary ~rtery disease cannot be s~ely re~sc~ ed. This subset ineludes patienk wrth diffu~e corona~ se~se in whom bypass surge~y is tccl,ric~lly not ~esslbb, and patients with preclusrve co nk~iJity such a~ severe lung 20 disease. In ~ese patients, bn~-t~ ene transfer to protect ~e myocardium a~ainst chronic recurrent s~hrn~b is belieYed to be p~rticularly effective.

Anlmal ~lodel of ll~yoc~rdlal l~ch~mi~
Important pr~requisites for ~u~hl studies on ~one therapy are (a) 2~ w";.titl~tion of an adequate animal mOdBI which 18 applic~ble to myoc~rdial ischernia of an P-.~GIrl~ous patient populati~on, and which can provide useful data regarding mechanisms fDr myucar~ial pr~tE~lion ~ the seffin~ of myo~ar~i31 kchernia, and (b) ae~urab evaluation of the effects of ~ene transfer. From point of Yiew, none Qf the pri~r art ~ ~atisfactory. ~t iJ, proposed in the pl esent 30 inv~n~on t~ use a porcine mod~l of myo~rdial ischemh ~at mimic~ clinical coronary arbry dis~ase. rLcE...ent of an arnero-~ c~r,~tricLor around the bft _1~

rr~ r~uOOll~J~ nr~Jc~J ~~ o~tooctlc ~ { r circumfl~x (LCx) coronuy arter~ results in gr~du~l cornpbt~ clo~ure (wKhin 7 d~ys of pl~oc...~n) w~th ll.hli,~l inf~rction (t% of th~ bn ventr~cle~ 1% of ~o LCx bed) (Ro~, et al. Cir~:vJ8~;0r7 ~2:177B, 1~gOt Roth, d al. Am J PY7ys~01 23~:H127~, 1987, Whib, et JL Ci~c R~s 71:14~0, 1992, Hammond, ~t ~I.
S Cs~liol 23:47S, 19~4, and Har",.~nd, d al. J Clin Inv~st ~-2Wf, 1~93).
Myocardial hnction and bbod nOw aro nonn~l at resl in th~ r~gion pr~iousb perru~d by ~e occluded utery (ref~rred to as th~ ischemic r~ion), due to co l~oral v~l de~clupr.~nl, but blood flow reserv~ i8 insufficient to pr~vent jBChCr;~ia when Inyoc~rdial ox~cn demand~ hcrease. Thu8, ~e LCx bod is ~ubject to episodic ~schem~, anslo~ous to dinical ~ngm~ pectoris. Collateral vessei dev~lopnlcnt and flow function reidtiGrship~ are stable ~thin 21 d~ys of arneroid plac~--e.n, and remain unchan~ed for four months (Roth, et al.
Cir~ula~on 82:1~78, 1~0, Roth, ot ~I. Arn J Phy~ 23~:H1~7~ 87, White, et al. Circ Res 71:14~0, 1~2). It has been document~d by 1elem~try that anuna~ have period schc. I ,ic dys~unction in the bed at risk throughout the day, related to ~brupt i..u~as~ in heart rat~ during li~Jin~, interruptions by per~onnei, etc. (unpublished dsb). Thus, the mode~ ha~ a bed with sbbie but inadequate collat~ral vessels, snd i8 subJect to p~liodic isch~ er distinGt advant~ of the rnodel i~ that there i8 ~ nonnally per~used and 20 functioning region (the LAD bed) adjaoent to an abnonnally perh~ nd functionin~ region (the LCx b~d), ~ereby offerin~ a "control" bed ~thin each animal.
Myocardial contrast echoc~r~ raphy can be used to e~timate regional myocardial perfusion in the ~,Q3e.1t inv~ n. The contrast material is ~5 oGn"~o~d of m~o~g,~5~a1es of ~AI~t~ and incr~~~es the ~oniaty ~whibnessn) of the ~e. The micro~gr~dl.:s distrlbute into the coronary a~er~es and myocsrdial walls in a manner that is prop~,lional to blood nOw (Skyba, etal. Cir~ula~ 1513-1521, 19~4). PJthough it b dif~iwlt to obtain p~c;_c quan~tative il~fv~ ti~ll w~th thi~ technbue, it has been ~hown ~at 30 peak int~r~ity of c~"~st ~ closely correl~d wi~ myocardial blood flow as me~sured by mi~ospher~3 (Skyba, et al. C~rcu~on 90 :151~1521, 1994).

lil--K 1~ '-b 1 b ~ ~ KUS:~b . N~ ~ ~t~L ~ ~1 J C~ u ~o~ ooc~c l ~ J ~
21740~0 Sin~ ~e ochoc~dio~raphic ~ma~os can ~ tely identil~ LCx bed, and myocardi~l collb~-~t ~hocardio~r~phy c~n be u~ed to ~~lu~b m~rocardial blood now, a hydraulic cuff oGcluder c~n be plac~d ~round the ~,ro;(i~ l LCx ~d,~ac~nt to the ~m~roid.
S PCR can b~ u~ed to dotect 5tress rolated hctor ~NA and mRNA in myocardium ~rom anim~ls that has ,~c3r~d gene tr~r~fer. In ~dJ:hun, ~o eeks ~r ~er~ tran~fer, myoar~ial sampbs from all fiYe lacZ-i~ed ~nima~s show su~s~ntial p~alactosidase activity on histolo~ Jti~n. In addition, u~ing ~ ~ a stress r~lated factor such as heat ~hock protein expre~sed in oe Is and in myocardium from anim~l~ ~at have received ~ene transfer can be dernon~al~.d.

EXPF~IMEN~ 1: Adenoviral Construc~s A helper independent r~plication defic~nt human adenovirus 5 ~ysbm was used. The genes of interest w~r~ and FGF-5. The fuil len~th cDNA
tor human FGt~S was r~ from plasmid pLTR122E (Zhen, et al. Mol Cel~
Biolo 8;3487, 1~88) ~s a 1.t kb ECOR1 rl~--,cnt which inc~ ss ~81 bp of the open reading frame of ~e ~ene, and ~oned into the polylinker of plasmid ACCMVPLPA which Cont~ins the CMV p,~"oter ~nd SV40 polyadenylation ~ignal ~anked ~y par~al adenovil~l se~uences ~orn which the E1A and E1B
genes (essenbial ~or viral replica~ion) had boen ddeteJ. This plasmid was co~transfect~d tli~,ofeotion) into 293 oells wit~ plasmid JM17 which contained the enbre hurnan ~denoviral S genome with an A ~i;tio.~al 4.3 kb insen makin~
~M17 too ~rge to be ~nc~psidated. Hom~logous rescue r~corobi.~t;Gi, re~u~ted in adenovir~ ~L~ nbinir~ the tr~nsaene in the absenoe of E1A~E~B sequences. P~though these r~c~-,binant~ were nonreplicaffve in ma,.""~lian cel~s, they could propa~ate in 293 C~lls whiCh had been tra,.s~l,l,~l w~ E1A~E1B and proYicled these csse~ dl ~en~ products in trans.
Tran~f ~d oells w~r monitored for eYidenoe of cytopathic effect whiCh u~ually d 1~14 days aRer transfection. To identify successful r~con~bin~nts, cell supcr~,atant from plat~s ~howin~ a CytO~idtlliC effect was ~a~ed ~rith ~1~

~ C 1~ rr~ ~u~Jl~1a~ ac~ u ~u~c~ cc -~
~174041~
pr~t~ ase K (50 m~ml with 0.5% sodi~lm dodecyl wlht~ and 20 rnM EDTA) ~t 56~C for 60 minub8, phenoUchlorof~ extr~ed and ~thanol precipitated.
Suo~ssf~ con~binant~ w~re then id~ntif~cl with PCR using pnmers chnlqu~s 1S:8B8~72, 1993) compl~nlentar~ to the CM~ ~,ron~oter and S SV40 polyadenybtion sequenc~s to ~rnplify the ins~rt (the expecW 1.1 kb ment), ~nd p,u.,6r~ (Biotechniques 1~ 872, 1~3) d~bned to w.,~.,Etantly ~mpli~ ~denoviral ~equences. S~cs~~1 ~c~,lbinant~ then und~rwent t Ho rounds of pbque pl., if ~60n. Viral stocks were prop-g~d in 293 oells to titers r~nging betw~n 10'~ ~nd 10'~ vu~l par~cl~s, and were 10 purified by double C~CI ~radient oentrifu~on pnor lo use. Reco,-~inanl adenoYiruse~ enGoding p gs'~ d~, or HSP70i were constructed using full kngth cDNAs. The system use,d to generate r~c~"lbinant adenoYinses nllpOs~d a packin~ limit of 5kb f~r trans~ne inserts. The ~nes propo~d, dri~en by ~e CMV promoter and with the S~40 polyadeny~ation sequ~es 15 were ~ 5 than ~ kb, well within the packagin~ con~aints. Rewrnbinant vectors were plaque purihed by sbndard pro~ur~6. The r~sutting viral vectors were ~r.,pagat~d on 293 celt~ to titers in the 10'~ 1012 viral p~r~cles ran~e. CBII8 were infected ~t 80~t~ confluence and harvested at 36 48 hour6.
A~r free~e-thaw cyctes the cellul~r debris W~l8 ~.cllchd by standard 20 centrifugation and th~ virus further purified by doub~e CsCI ~radient ultracentrifu~ation ~discon~nuous 1.33/1.45 CsCI gradient; oesium prepared in 5 mM Tris, 1 mM EDTA (pH 7.8); ~0,000 x ~ (2 hr), 105,000 x o tl8 hr~). Prior to in v~ inJection, the viral stocks wer~ desalbd by ~el filtra~on through sepharose columns such ~s G25 ~ephadex. The r~sulting ~iral stock had a 2~ finsl viral titer in the 10 '~-10'Z vir~l parU~ n~e. The adenovir~ construct was hi~hly purifed, with no wil~ Jot~ lly replica~v~) virus.

EXPF~IMENT 2: Adu~t Rat Car~iu."~ in Cell Cul~ure Adult rat cardiomyocyt~s were prepar~d by Lan~endorf pe~slon with a 30 colla~nasecGn~i.,in~perfusate~wordin~ ,Jmethods. Rodshaped oells wer~ cultured on bminin coated pla1~s an~ at 24 hours were inf~ d with .c ~ J r ~ J~;U~O~ r l~_r~l~r~_r~r~JC~J C~ I ~C_I I rJ Ir~ ;OOC~C I ~ ~ . CJ~ Jl 217~0~0 ~o ~alado~id-60~nccding ~denovirus obtained in the ~boYe Experunent 1 ~t a mu~plicity of inf etion of 1:1. A~er a fur~er 3~ hour p~riod the c~lh were nxed with ~ r~ldehyde ~nd incub~tod with X~l.
Consistently 7~90% of ~dult myoc~ ed the p ~-~ncto~id~se 5 tr~nsgene Jtter i-~ with the r~n~binant ~ ovl~Js~ At ~ multlplicity of intection of 1 2:1 th~re was no cytctoxicity observed.

F~PERIMENT 3: Pi~ ~yocardium /n V~
Th~ p-galactosWa~ in~ adenoviral vector obtained in th~ above 10 Ex~r;lt~nt 1 was prop~g~te~ in ~.~l~issive 2~3 oells and purificd by CsCI
~radient ultrac~ntrifugation with a final viral titer of 1.5 x 10'~ viral par~ioles, based on the pr~c~dureo of Lx~crin~cnt 1. An anesll~Li ed, ~cntilat~ 40 kg pig underwent t~,or~c-Jtomy and ~l~tion of ~e bft circumfiex and le~ ~nt,,ior descendin~ coronary ~,t~.ic~. A 26 gauge bu~orfly needle wa~ inserted into 15 the mid bft anterior Je_~nding (LAD) coronuy artery an~ ector (1.5 x 10'~ viral pa~cles) was inj~ted in a 2 ml ~olurne. The chest was closed and ~e animal allowe~d to recover. On the fourth post-injection day tile animal was sacrificed. The heart fix~d with porfused glutaraldehydel s~or~d and incu~ated wi~ X~al for 1B.5 llours. After imbeddin~ and ~C~Ohil~ the ffssue 20 w~s counterst~ined with eosin.
Microso~pic analysis of tissue sections (tr~nsrnural sect;ons of LAD bed 72 hours after intracor.,~ y inject'ion of ~denoviru~ containin~ lacz) revealed ~ significant magnitl-de of ~ene ~ansfer obs~rved in the IA~ coronary bed wit~
many ffssue ~ections dc~ 8tr~t;o9 ~reater ~an 50~0% of the oells staining 25 po~itively for ~ ;tc si~as~. Are~s of the myoc~rdium remob from ~e LA~
c;rculatory bed did not demonstra~e X~l stainin~ and sen~ed as a ne~st~ve control, while dfflu~e expre~sion of ~ ~ene was 0~8er~ed in myocytes and in ~otl,~lial oells. Th~ majo~fly of myo~s 8hc~ ctosirln&~ ~ctivity (blue stain), and, in Y~bse~ tudies using closed~:hest intracor~nary 30 i.l,e~t;cn, similar activity was pr~ent 14 days a~er ~ene transfer (n~). There was no evidence of in~bi.,m~tion or ne~osi~ in the areas o~ tran~ lion.

~1~

~1'~ lC ~ ~ ~ rr~ ~VI ~O ~ J ~ nr~c ~ ~ r ~c ~ l ~ J~ r . c~
21710~0 klME~T 4: P~ Con~ ti~n Model ~nimal~ ~nd In~umenhffon Petail~ ~re based on pr~viou~ ~dies (Hamrnond, et ~I. J C/in In~es~
92:2~ 2~52, and Roth, et ~1. J Cl~7 Inv~st 91:~3~ 949, 1~93). Anirnals includes 14 do~"~stk pi~s (3040 k~). A le~ttho~cotomy is p~.. f~ l8d under sterile cor,~lK~n~ for ins~",e.~ ~n. Ca~et~ are plac~ in ~e bft atrium ~nd aorta, pro~iding ~ ns to me~sure r~ional bbod fbw, and to monitor pr~ssures. V~fires ar~ ~Inured on ~e bft ~trium to permit ECG r~cor~in~ and atrial pa~ng. ~inally, an ameroid is placed around ~e p~Ailll~i ICx. Af~er a 10 ~t~ble degree of ischemb has de~eloped. ffle tre~ t ~roup (n~) r~ceiv~s an adenovttal cons~uct that include~ genes for ~ISP70i (a heat sho~k protein), driven by a CMY ~Jrol~obr. Conttol animsls (n-5) r~rv~ gene tr~nsfer ~~vith an adenoviral construct ~at includes a r~porter ~ene, lac~, driYen by a CMV
promo~r.
1~ Ad-novira~ Con~truc~
The helper ind~p~nde.,l replic~tion defic~ent human adenovirL~ 5 ~ystem constructed in the abo~e Experiment 1 b used. The g~es of interest are laeZ
~nd ~SP70i. The ..,at~nal ~"e~ n ~7YO is highly puri~ed and c~t~i"s no wild~ e~ tiG" ~mp~t~,nl) adeno~irus. Thu~ the possibl~ in vivo 20 adeno~iral in~e~lion and innan~m~ tilb~ion in the hean are minimked. By injecbn~ the materi~l directly into the lum~n of the coronary artery by coronarycatheters, it i~ po~s~le to 'target" ~he ~er~ ra~er e~fec~vely. Gene e~r~ssion when deli~red in this m~nn~r in minimal in the liver, and ~iral RNA can not be ~ound in the urine at ~ny t~me after intr~co~"ary inp~ion.
25 D~liv~ry of th~ Tr~n~g-n-One of ~e present in~ont~ k ~xpo.i.:,~ in iar~e animal sur~ery(Hammond, et al. J Cl~n In~sf ~2:2B44-2~52, 19~3, Ha~ ,o~J, et al. J A~ner ColJ Cu~iol 23:47W82, 1~4, Ro~, et ti. J Clin /nv~st 91:~3~ , 1993, and Ping, et ~I. Am J Physiol 267:H2079, 18~4) and with coronary arlerio~raphy 30 ~hum~n~ and pi~s). An angio~raphy lab~r~lo-y for animal st~l~ies is ~vailablein an adjaoent labo,~,~r. Inj~ction of ~ construct (4.0 ml containin~ 101' viral _1~

~r~ LC ~0 10 . _~ ~ r~ r~u c l l 1a c~r~ r~oor1r~a~La c~u r~c7 l u lcu aDc~C ~ r 217~0~3 ~rffcl~ of adenoY~ de by in~in~ 2.0 ml into both the le~t snd ri~ht ooronary artories (oollat~r~l tlow to the LCx bod appeared to corne from both v~els). Anirn~s are Jnes~tk~d, and arterbl ~ ss aoquires ~ Ule right caroti~ by c~n~own, a S~ Co~lis ~ath ~s placed. A SF Multipurpose (A~) coronary c~theter is u~ enga~e tho coronary ~rteri~ Closure of the LCx ~meroid i~s cvn~-,..e~ by oontrast ini~ion hto tt e k1t main c~ artery.
The ~th- ler tip is then pbc4d 1 em within ~e ~rterial lumen so ~t minim~l material will be lost ~ the proximal ~orb dunn~ in~ction. This procedure is carr ed out 1'or ~ach of the pigs.
~e~ment of ~yocJ l ProtecUon The strat~gy for myoc~rdi~ rok 1i~e studies hcluds the timin~ of ~n~gene delivery, the route of admini~tr~tbn of ~ t~dl~s~ene~ and choice of the St~88 related ~ene, U8ill9 the .f~,rili6~id oon~ruct including a reporter ~ene (bcZ) ~nd that including a stress rebte~ factor ~ene ~ well as ttle aforesaid pig models. The ameroid model of myoc~rdial ischemia k ~l~c-~e.-, and ~ene trans~sr i~ performed a~er ~able. G~ne tr~r~fer ~re ef~ectecl by intr~ro"di y inj~tion of the viral oonstruct several day~ priior to non cardiac surge~ or a diagnosffc cardiac cathet~,i~t~on such m~ bvels of pr~te~ e stres~ factors in the myocardium will be hi~h dur~ng ~e ~nticip~t~d ~ gery or percutaneous 20 revascularization. In ~Jition, ~ene l.~..s~cr by it~tracoronary inje~tion is conduc:ted at the ~me of diagnostic car~iac ~II,¢t~riz~tion just prior to 8ur~ery.
I~y~cardial pl~liGr, can b~ ~~ss~ J by the aforesaid echocardiography and mic~ OpiG analysis.

Claims (18)

1. A method of providing myocardial protection, comprising:
delivering a replication-deficient adenoviral vector to a myocardium by intracoronary injection into the coronary arteries, so as to transfect non-dividing cardiac myocytes in the affected myocardium, said vector comprising a transgene coding for a stress related factor selected from the group consisting of HSP70i,HSP27, HSP40, HSP60 and the adenosine A3 receptor; and expressing the transgene in the myocardium, thereby raising the level of stress related factor in the affected region of the myocardium.

2. The method of Claim 1, wherein said patient has non-revascularized ischemic heart disease and said protection is desired during planned non-cardiac surgery, wherein said vector is administered a plurality of days prior to the planned non-cardiac surgery.

3. The method of Claim 1, wherein said protection is desired in anticipation of complex percutaneous revascularization, and wherein said vector is delivered at the time of a diagnostic catheterization a plurality of days prior to the revascularization.

4. The method of Claim 1, wherein said protection is desired in anticipation of complex cardiac surgery, and wherein said vector is delivered at the time of a diagnostic cardiac catheterization.

5. The method of Claim 1, wherein said protection is desired in a donor heart to be transplanted into a host patient with a coronary disease, and wherein said vector is delivered at the time of a diagnostic coronary angiography prior to explanation to rule out coronary disease.

6. The method of Claim 1, wherein said protection is desired in a patient with diffuse, nonrevascularizable coronary artery disease, at the time of a diagnostic coronary angiography prior to explanation to rule out coronary disease, wherein said vector is delivered a plurality of times.

7. The method of providing myocardial protection according to Claim 1, wherein said transgene is driven by a CMV promoter which is contained in the vector, and said vector is delivered in an amount sufficient for transfecting all cell types in the affected region.

8. The method of providing myocardial protection according to Claim 1, wherein said transgene is driven by a ventricular myocyte-specific promoter which is contained in the vector, and said vector is delivered in an amount sufficient for transfecting exclusively cardiac myocytes in the affected region.

9. The method of providing myocardial protection according to Claim 1, wherein said vector is delivered in the form of a viral stock having a final viral titer of

10 10-10 12 viral particles.
10. An injectable adenoviral vector preparation, comprising:
a recombinant adenoviral vector, said vector containing no wild-type virus and comprising:
a partial adenoviral sequence from which the E1A/E1B genes have been deleted, and a transgene coding for a stress related factor selected from the group consisting of HSP70i, HSP27, HSP40, HSP60 and the adenosine A3 receptor, driven by a promoter flanked by the partial adenoviral sequence; and a pharmaceutically acceptable carrier.

11. The preparation of Claim 10, wherein said vector is in composition having a final viral titer of 10 10-10 12 viral particles.

12. The injectable adenoviral vector preparation according to Claim 10, wherein said promoter is a CMV promoter or a ventricular myocyte-specific promoter.

13. A method of production of a viral stock containing a recombinant vector capable of expressing a stress related factor in vivo in the myocardium, comprising the steps of:
cloning a transgene coding for a stress related factor into a plasmid containing a promoter and a polylinker flanked by partial adenoviral sequences of the left end of the human adenovirus 5 genome from which the E1A/E1B genes have been deleted;

co-transfecting said plasmid into mammalian cells transformed with the E1A/E1B genes, with a plasmid which contains the entire human adenoviral 5 genome and an additional insert making the plasmid too large to be encapsidated,whereby rescue recombination takes place between the transgene-inserted plasmid and the plasmid having the entire adenoviral genome so as to create a recombinant genome containing the transgene without the E1A/E1B genes, said recombinant genome being sufficiently small to be encapsidated;
identifying successful recombinants in cell cultures;
propagating the resulting recombinants in mammalian cells transformed with the E1A/E1B genes, and purifying the propagated recombinants so as to contain the recombinant vector, without wild-type virus therein.

14. The method of Claim 13, wherein the to a final viral titer of 10 10-10 12 viral particles range

15. The method of production of a viral stock according to Claim 13, wherein said plasmid into which the transgene is cloned is plasmid pAC1 or plasmid ACCMVPLPA.

16. The method of production of a viral stock according to Claim 13, wherein said identification comprises the steps of:
monitoring transfected cells for evidence of cytopathic effect;
treating the cell supernatant from cell cultures showing a cytopathic effect with proteinase K, followed by phenol/chloroform extraction and ethanol precipitation;
identifying successful recombinants with PCR using primers complementary to the CMV promoter and primers complementary to adenoviral sequences; and undergoing two rounds of plaque purification.

17. The method of production of a viral stock according to Claim 13, wherein said purification comprises the steps of:
propagating the resulting recombinants in cells transformed with the E1A/E1B genes to titers in the 10 10-10 12 viral particles range;

purifying the propagated recombinants by double CsCl gradient ultracentrifugation; and filtering the purified recombinants through sepharose columns.

18. The method of claim 13, wherein the stress related factor is selected from the group consisting of HSP70i, HSP27, HSP40, HSP60 and the adenosine A3 receptor.