- Email: [email protected]
Muscle-based gene therapy: realistic possibilities for the future
therapy'°
review, ~ ~ of ~ ~
Lh~~
~ ~ m~ as a target tiss~ ~or ~ n e t h e ~ , d ~ s ~ ~h~ that have ~ used for m u s c l e - b ~ ~ therapy, s u ~ ~ ~ di~se.~N~ using ~ and discuss some of ~ e obstacles th~ remalr~t~ route to muscle-rinsed human gone therapy, ~n~fia~ ta~eI tissue is e~e ef the cri~ica~iss~s 'd in desigNr~g a geee-theraW a ~ a c h for ~ y ~, tl-e choice of t ~ e t tissue is dictated by the he ~ a s e it.lL For e~ampte~ i~ is get,orally ay eNthelium mus~.N targe~ ~ g~ ~cessNt : fibre:sis. However, in m~y Nsea~s IN a~es o e ~ o r ~ N a s~cific cett tyN+ a+ we~ ~+safe~y criticN de~m~i~ants of the r~os~ iaN ; a ~umber of p~N~er~ies~hatmake i~~ attractive ~n ger~e ~herapy°First, broth ~eo~atN a ~ ~ult ily i&mifi~e and isolable s~emcell ~bx sketeta| myobl~.s ca~ N growr~ ~o I~'ge numbers i~ ~blytra,'~Nuc~ with rezombie~nt genes using a , including retreviru~s, liNfecfi~s and. DNAm~dified m inlo lll~fi~|.fatecJ d~ (i°~) ~njec~ier~ s~ch gene~icaliy myofibers te Ncorae stably i ~ sue. ~ N ~ k ~ m~ified ~ pr~e mrs g N~ins N vicm argdM vi~v, ~upp~yN skeleN m~cle Women,s the e~cien~ ~eim m ~ systermcci~cuIatienoNne~ly,~eNa| ~ne m ~ o w or va~ular e~dotheliN ~Ii.s,has a N N that heretically medifi~ mym~berscan. if rgicM|y m ~ v e d with,~utadverse com~equences. ~ t ~ i ~ d on ~ proposes ~ demons~ae that ~ y o b ~ can be u ~ b~Lhm produce musc|e ~ m s ~ y deliver physiologicallevels :rues and cytekines t(~ the systemic circulafief~ n m the Nv~ages of sketeta| myob|~s~s for | rau~le ~e rare ~ N t~ng ~press DNA ve,~rs fo~t~wNg dh'ect mpeny ha~ recently ~ exple~d m deve|~ that may ~ useful f~r a wide variety of iw us~ N addition m its obvious utility as a ~get erapy of iN,orbed my~aNies, ske~eN rausc|e
i
i~
7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a
Iegs, liea~ior~-defeetive
Ge~e~ic modificatio~-s
myobtast
Plasmid
M~s
~
,1~ I',
/ Myoblast hawest
e
b N~ob/ast
/
',,4,
il;
i.m. iniection
t
i.m,. iniecSi:on
iota. iniection
!: iI
Fusion
\~ I ! I
Skeletal m u ~ I e I
,
il
Unmodified mydibe~
Modified myofiber F~:~ 1oA ~hem~is ilusIra£io~0~Nf~ sIraI~icm {or m~acIeg~e lt'te~a~y.The sIeps i,~woIvedi~ the use ,o~(a} my®last transplantaSors it) ~epIcaIiom @(ective a@s/@dmta~4 (e} ~NA for m ~ - ~ d @r~sfsem~:yam @pic~o
is a~ excelent target {issae for geee therapy approaches designed to treal inherited aed acquired set,am protein def}c~encies and fbr genebased vacciees.
Ex vim and ie ~im ge~e ~herapy appr~aehes iarge~li~g skele~| mu~le of
~ e first m u ~ 1 or
a|ly
tN~r fled m
im;o|ved ~he h:m b ~ k into host ma~le
by i.m. i!ieclio~ (Fig. I: Table I)'~~:' ->:( In these sludies, normal cuII~red ~yobtast~ or 11~ .c~!:iqt t r a i l . ] i c e d i/l ~ifft)" w i t t replicalion-
defective re~mvir~s~',s or p=asmNs cX~+eSSir~gtie genes enc(ydieg I3galac~osidas~e, humae growth hormone or factor IX were reimplamed ieto sy~gN~eic mice by simple L,mo iriiection, By his~ehgical analysis, such/eeetically r~:x_tifi~ myobhs~s we~e shmvn to NEe wi~h ee@ygenoas ~ m s~ab~y ime ~he bos~ m ~ l e , i~ N the Lm. i!jection of mladvely se~ll eu s of such ge ly m / i t i e d myoblasts ~s~Red in Ihe ~ec of
167
my~Nast tranffer is a.n inhe:en~ly~ab~.>~oi~teasivearid e×{~:~sivepro cedric+ rwq~iringeither the is~>htionof pfma~~+Wob~a~s~Lrom cacti A t t l ~ h L ~ initial studi~ provide:l an important pr~.s~fof pfi+> tremied patent or imnveam+uppmssio~ ~bllowing m>nau{olegems myoNast woa~spka'*mtio~.Second, human m~'~e appear~ to ~ sign ci#e concerningL~ feasibilityof using ~ M and ge~edcaHy~ i + f ~ m y ~ h ~ Ns the gone therapy of ~ h inheritN myopath}esand aificmstly~essreceWiveto t~:~nsNa~tedmyoblaststha;e mnu~: ma+;c}e. Thm, re:+ + feces ~ s N myoNast tr~ms~er ~?~' to g~la~: ~ s i ~ mc~a~fut human g~rg therapy:.Fi~t+ cant a~t N ~ 6 × 1~ celi recent r e d s have NN myobhst N :~e~ts ~ y N e~anced by No-injection ir~j~ of fie gm~cle, dm~ fi~ngs remain + a~d it is ~ t ctear fffat they are app+ke&le m human therapff, h summon%akb~gh n~yoNas~~aasp~ was historicaJly irnv~am in d~elopi~g ma~te+~sed gcme therapies+ i~ m ~ ~ ~ matnxo Thin ~ n m mutat~ in is unlikely m play a major ro~e is hmm>ange~e therapy ~nless it is ~ssible m devek~ a uuiversN dc.n~rmy~L';}asttha~ca~ ~e efficier~tly engrafted into h~man muscle+or unless it is ~0ssiNe m develop tram+ ~ ~ (~ - A p~e~n ~morm invotv~din r~u{ming sieur immun~ppressive ~egimens that wrmit tor~g+termmy~{~t graft survival in huma~ mu~leZL - ~n~ contextof tN~ r ~ i ~ , the attacbmen~a~ e~tty Dimc~ DNA injection In ~ ) + ~\Nff ~ d co+w(wke~ made+the surprising th~ skete:~l can take up ~ d s~ab~yexpels im~ma~u~gdy ~ ~ ~ r~R ~ t~ m ~ ~ fe,~mgnDNAin~ a injec~M ptasmid DNA~, 'tNs finding r:d~d the ~ b i t i ~ y of uskg e~tL direct DNA injectio~ as a straightfem~vardmeth~ of M vivs gone - MMum+~ ~ + e ~pid~ t~at faei~iba~ the tranN~tN~rapy in m~ele. Direct DNA inj~'tion has a n~m~r of ohvir~s adwavmges as a gone tra~uction a~oach ( ~ b k ~}. F~rsL k is a simple in vivo gene transfer tectmique that does ~ requi~ ar~ iNi:cL~om~ ~ - ~ enzym~ that ~s p r ~ o m ~ y ~eal+z~ tkms vector m~di:hatis feud@ applicable m large s~m~m of ~es~ts Ptasmi&based vectors are easy to constrgct, a~d +he prepm~do~ and ~ of e ~ t ~ r proteins+ ¢ + # ~ m t e m ~ ~pi~,+ storage of large amusers of DNA is relatively swai + aMitiom the a~am~t of traasgene expressio~ is progerdo~al to the ~r~ ~ ~ ~ ~ain~g a r~r~ @pFK~phm~ re+tuber of DNA injegtions, and ~ a ~ e d injections c ~ ~ easily ad~thohym*mtegroup ~ d f~ta~es the t m n ~ t of tlmse protein~ mieisteredV2:( NnaRy, transgene e×pfessio~ aN:marsto be s~hle for perk~Js of at ~east 12.q9 momhs Nthd-,M~gdirect DNA iNe.ctio#:o - ~ stem ¢et~of m u ~ ti,,mm,ff is m~non~lear a ~ Despite these advamages, the usefialnessof' dir~xt DNA i~ectien has ~,~}~. It c~'~~ d~ffemnt~teto form new my~be~, or fuse been gready limited by the finding that the e~$ciescy ~:ffgone transfer folk~ing Lm. of pt~smid DNA g extre~rely lea*: ~ss Lha~ 1% of the myofibeesi:e the ~ of iv~'ecti~rtm~e up ~ d e × ~ s t ~ ptasmid DNAm DespRe ex~nsive vaxk by mmqyg~r~ps, it bus th~s *~ been m i~rease significantly~ of bg vivo gone ~aasfer fol|owmg Lm+~nject~ N DNAoH~'eve~; recur s~ad+ ins have s h ~ n that ~ |evets of t geae e x ~ s i o n l/oh lowing direct DNA injec~i~ cam be incma~.l at le~gst5(Lfo~&by i~cw~ing firm tran~ripfiona~ efficiency of the iffWaeaJ p~mid :?. Ti~e~ fin~ngs, N~g with ongoing studies designed m ~+v+,o}. mRNA stability and tm~sMional efficie~zy, a+ weii as to increase transgene or mI~NA c ~ numbers Mlewi~g direct DNA injegfiom T ~ - The +nlm~aO&mof fore~ ~ A ir~o mils in eel+ hold prmni~ for exparding the usefulness of this approach. Howevee, at ~ proses time, d i ~ t DNA i~jection ~ a r s m ~ u~fu! only -A ~ ~ h=.~b ~ ~tab~i + ~ intoanother for the development of D N A - b ~ vaccines that require lOwolevel ~ d wa~sient~ o m b i ~ t g~ne exp~ssion i~ n'm.~le (see below). T ~ ~ d ~ # - Th~ mr+ at wt0~g~a specific mRNA i~ Aden~wims r e c t o s R~qplicafiono~f~6ve ad~novini~s (RdAd) are one of ~e most promising vectors for in vivo gone d ~ a ~ in skele~ m~vAe+Adeno+ im eell~, an~ IN~mid~ are P/p~.BI viruses ~ douNe-swandedhuman DNA viruses that typically cause veer+ ~tf4imited res~xato~ tract ivdecfions+ RdAd in which ~ El ge~s have been replaced by a p~o~er-transgene eas~tte can effficiently #~ysio~%icd ~vels of ~ombiuam prmeins )9!9the systemic ci~u+
m
isthct ~(x~{ replicadsg ~d ~?o:~>~::plicair>g col types in v~v< b~£ ~ack the ability io geoOoe week T w o months em~e i@ec{~ous ~ g e ~ y . Tte abi}i~y ~0 isS:'c~ ~or>replicai~g cel types is ~'ticaAarly imo p,or~a~ fbr i~5~rive m~1 m{I wete Lm. wif)h I x 10'~ 1 ~ 9 t~IVa d sy circ~fior~ of i ea~fiei/a~t mice .~£c;Z,a gdAd t~ ~vi~@ of ~e :asZ~ A#~ o ~ w ~ ~ Va~ , ~a were , as4 fo~ %.r a leas{ e>g mo,nNds o te th;eir grea (~g ae Nan}. @~ ~ a a'ler Lm i so, thee ~.se of RdAd for ma~:le(a~ b} N2@ Of mi~ h}~NNVCJ5~ ~ its ~Pffdf S. gene t Ms bee~ ~imi{ed by the fi~disg g£~r t>~o (¢ d:} o~W~e SC/O~ce tea~ 4 I@a~.~ o ~ s e Z N a ~he use of t~ese veaors i~ adllt i~m~uloc hosts p r ~ c e s (rely {r:a~sient mcombi~aN gene expression i~ vivo (lasting 2 4 weeks) (Fig° 2). veto it has p;roved imp£~ssiNe ~o re- skde~at m t ~ b has beee shown {o r~.ut~, {e Fdaive{v, bw tratsd~cadminister RdAd s~ccessfuly following a~ inidal i~@c{km° Recem tio~ efficienciesC Ade~o-ass~>giaed virus (AAV) is a [email protected]'ar@d DNA vires ~ha c ~ i~Ea ~ah dividing and aon~ividi~g ~lts~ st have e ~a ~s~ ~e and d ~ s no~ m c a ~ human disea~:ZsoWhib wtd-VN AAV r e s ~ s i N e N~I for {he ie~ of exp~ssie~ ob ; ~ I% tea}rubicon{ AAV wi~h RdAd a~d for fie i~ili~y m iNster vim¢ e'~'~,C ~i¢ i~{egraNs s~ifi~at:y a a site on t~ks ~Ns s~ifici~y of inmgrati~L if~creasing the risk of i=~rtkml T~el ~ed b(~& R~eig~ m~gmsesis, t~ a~itioe, i~.is diNe~lt ~o preNfe Ngho~ler ~zombi° a~d iral d vim> arM. r~sagiAAV. Nnaly, ANV c ~ acc~m no ~gre th~ 4.7 kb of sLab-term e×~essb~ i~ vireo S > izi~g foreign DNA, gheNby limidng its ~N|reess for the treatmen~ of pred,& virat o ~q~in~g the expr~si~ of l ~ e cDNAs. Th~s far. on ~ i f y i n g ~he s ar~ ~ viral a ~ {m hmr~an di deve ~vei ~ hold fNfeedns have: mx proved m he e s ~ i a l / y ~seNl fbr the s~aNe ~ran~:acti~ of te~mi~aty differe~dat~ skeletal myofi~rs in vivoo for y the of in H~ever, ~ ~ t @ v d @ ~ a t of ~w. ~ , e effident iiNfecfins and based gene In on, k wil le to d e v d ~ ring sNcifi¢ eel ty~s by integrating viral for the sy admi of RdAd m s mu~le the Nssibiity of celM~ proteins into lipesomes may N>M promise ~br fie Nvare if t , ~ are m he useN~ in the of ir~berited deveiopmem of t ~ f ~ t i n - b a ~ d mu~le ~a~Nucdon sys~emsX'o myopahies, s~achas DMDo
8
Other vector zvsgemsofbrmus~@gone ¢kempy @her are as for m ~ t e gene l)o bare ~ = used m y in ~ a~ m~ for tN in rive of skeleN my of their iiVy m non skele.~l . i ~ ,
even
d
r~Jr,ises
y into
Di~a~ ~ fee ~ e b ~ o m u ~ J e o b a ~ d gene theraD' lar ies The m ~ obvie~as ef mu~ g.ene is ~ t of singlerecessive my , such as DMD~. DMD £ an X fnked my y tha ~fee{s ximaely 1 in ~as. ~a is by m of %e dys ge~, ~ i structural protein the has been 169
///
/
~
~mphm geac ~o +go e:dm+~'+e~y~a%+ee~+~e<'le
"
+Wa,ss Of (+*¢ h+a+'+a+o+~g/a+:++sm+++(}+~+s+++~+¢m~'}oP
¢ba+le~ge +~ rise. ge++e ff~ec@y of f~+g'~eri~+4 is ~hc @~eR+~em of a s~s+emi:,+:
++//
s~ei~k~ty
~mi~aJly ++~iaed myofi~rs i++ a m++veb+ + g e ~:+gam+++smsmeh am~rm+L
@
k l +m+m "..
&r+w+ p+vge#+ de~++:iencas A~+}~m~ ~+ mm+++a @m+¢+pi+msve+ +.at t++am~ab+echat+eliges +~J+ef+>~:++veger+e
++
f
@4Jaf+o~
a++++++++}++0 +MY++++{+~++'++++~-~+++'++++ ~m p+J+~+C+++g+
a ~eta~ve~y la,ge mi~@er of ~,eam ~ + @t++eie++¢+ a~+a~ ++ ~ we}| ~¢',,~ tO m++ag+.+.> ba+d gc~ +++g+u{+icstTabk+2}+¢+~+++ ~+++++'>+++ As @s++i~d +{~:+ve,myo+++i~+mca+ c a @ ge+m+~k+a~}ym~×fifieda++ cam efMde+~t+yse+ ~%m+.,
,
¢+Fe~¢
J~ + "-+... +%>_ +,++
+ TgPa++ ~g
+ + + +a~{++zmim tBempe+ti¢prc> te~m s+¢h a+ E++ m+,] f~++tO++ +X+ tJ+a~a+e ++X b~++a++, ++*em~i from myofi+vrs g)dr+ Pe+~ +++:+st++t+m+++emit c + +++:+,~t~m~+~a++++y+ m of a +e+mmpro+ m++~+eik+e~+:y is ++++h+im+a+~y+++¢h ++mp½r @+a*~ ++:~_.:+~+¢t{++>+~ of #+f+i++++eri~edmyopa~}~y ~ m + s e +~ig~+m++k' ~raa+~+te~i+meffMe+++ c++ am +~:o~reqm++J + o~+y a m+aivety sm++li
+',..
m++k ++eed+ ~:+~+ +++~+p+++temof +m~em+~ ++a+:++kvem~++:g~+m~++:e~++~c~ g ++++
~
~
++:e+.+~+ + + m
+iag+ i+m++r@cti~+++of R~Ag e+++c++i%E+~+
+++
@+ m
RPaA& T+P~ i+i++di~gsmg#,e~+~@m C~ Lm+ ~' RdAd h+++~+J~+ ? + + may ~ m~+ f~+ hu+~+m+ di++ad ~+i+s im+he +++ from+e+ A +r+~ Lm+ imp+m+ ~ +++~ of a p~+m+d+,b~d E+++e~+:~+s+ skin v+t~+ ~+ +a+ t++++++h+m++m +++it m kmg+~em~~ + s+v++t~+{s i~ m + (T~+~+ S.K+, Sver+~m, EC., BiaS+ ~.+ m+ ~+i@m+$M. LA h+a+rmm wo+#+ ++R~ait+ethe kin+ i++jeco i+~+
~ amJ t ~ +¢++ Lm,
eff+k~b:s of t+se p~a+m~ +++'+~+may dMv; t k +++ of m
i+++++++>+~ ~ m ~
~ ~+ t~++
~+e+ my~s~
~m ++f
+ ++ m+ +++f+ mi~ DMD+ Mos,
+a ~ m + Proem+
grea
+ / ~ Lm+ {R+RdAd a ~ d ~ { DNA ft~ ~ m ~ ~ y : i+~saes~ema~i++gm
~k~
' . . . . . . . . . . . . . . . . .
~
e, . . . . . . . .
,s,
~
.
.
.
.
.
ge~e e:,,~/'essk:< h@>v, i~~:~dee¢~ ,DXA
ex F~aHy, t
sio~ ~
a%' h
dm~ca~ ~als are,~.~derta%er~,.
hes
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
p>:~e m t2>e~" pr,~d~.~c~.~:a~>f hc f ,.r<{@<:~pv~<~,S,~,, ed ,,~de ,:,>p~<>d~!:7.0e~:~ mr~:::~.',:~<:><:.pc<>< <> '~,: ,,,,.~,eX, ~I< :~ .},o <,,::~ ~<:,.::<.~:e i ~e r~,@:.:~:qu red: a~:>d4 : ~.,~,u:.,?< 'h,: expn:,<,,.o~: ,. ~o~ required. '7%~.>,.~~ h~:<%, c~.~m:>: D;",,.~A ;~ec or
kaHy i~ tica~b:' ld be eva~ ir~
~%d <~i>~a~ ~
~s
@s'os
: s:ore~ge ~,;es hg~fhed ly I s~ div, ases (LSDs~ a'e a ~hkd class of di rs {ha may ['~:, ~qie~larIy well s~hed ~o m~.>c[e-di gr~e gqe . ]£-ese rare ~'~:cessivedi s a e e>:h ¢h~vc{erized h} ~he ah~em:v of a sisgle ~%~w{k~>~:}a~~ysesem;.~e~zyme ~ypicaHy rc:d~-. ir~g i~ the [ym~,m~ai acc~am@aio~ of the s~abs~ra{e ~a,u~o~'sor,,of dse mis@g or defhctive er~zyme, a@ @timae[y ~ea@qg {o ceHs/a dys.Nr~(:~ioe a~(i:or dea{ho C~£~icaH> @ese diseases presea wi~h mema~ re~ardadom f?om ~e~roea~ dysNm:'tio~:~,a~d e~a~,.#mest of the lh,er aed spleen, flora accommodationof g~ycosamineg~ycan s the£: orga>. Mo-s~ LSDs resah m m ehiid Them ~e c~rr-e~t[y so e~.ctire ~eatme~ts for aW N {he ~SDs, makhag eea: &sere the ap., higNy de@:aNe, The s sNl ther@y of LSDs wilt ~:~ost prot~hty ~q~,.~.h:e tha a Nzge ~age of cells i~ a ti {live< @eem eee{rJ em:~'o~s sy (CNS}] ~ceive the reph~cem-eg~ e~,zyme, ~xt~mae~y, h~eve~; ly a~ eraymes ~:~ssess a tageti~g sigsal,, ma~ g ae, ]~a~{s,cd/s ~avkir~ga ~¢:ific ~yso,,oma~e~:~zy~r'~eear~ u~a~ ap e~:~zymefrom {~:e >e,~mmvia their mamm.,:e 6-phosphate recep~@'s a'~d delg<~r this eezyme m the {y~:}mes~ {ieg the/ysomma/ deficiP~ <~kAs ~ h:~Fig A a viaNe thera[~:0~{ics~ y w~:m~dN h} mmN~x:e m~c[e cells wi~h @e ge~e e~c~ir~,g {he/y:,osomal eezyme of igteresL dh'ec{i~g {hose cells to pr~ace high ~evds of e~yme h~ ff~e serum, which co~d ther~ N taker~ ~@ by cells m the [[~ea ~;,pleee a~d CHS The hw:dks {o t~: overcome h~ the corrccfi@~ of ly~esoma~ ~omge by ~he are, [~ ~ru'~ csa~s, similar m de~: for k> the for ~z~{~m.o ies° Fksh Ngh s of ir~a~t expre>
sio~ N ~ d h~ m a c[imd , ~d, ~his e~ km m~st N mai~ai~d lbr ~he ~ife of the h'@ivldual, ly, amt m{~t i ly tbr di char~:~erizedby' , ~he r~" /ysosomag eezyme cross tN °~ai~ i~ m tN CNS lities of di ., less, tb~ ~gem ckmiN of the genes resgmo for most N Ne LSDs, aa~dthe avai|ability of N{h smalb aN Jarge-a~imd m~. e~s of the~ di~ea~s, shouM facilitae the deveh@o me~t N ~oelieiea/NNs tN/izi~g myob|ast lamrafio~and i.m. i~kc~io~, of RdAd or Nasmid vc~:tors.
ge~ ape es. ~I2~e f;easfbiH~y of ~si~g d
DNA i
as a v~cc
tie~ ~.'.r wi@ as exp>essk~q p}ee,m}d c.es i ~J@>...s ~ith h~@a~doses of isfluc~> ',irus, S~b~.q~,:'sSy.
p% h~,u'
t < > , isch.@h~g he.rpe~, '.-. ~k~:'I,, I.ha @~e} '.,~h~"ge<,de w~ccesf@ m~.>:c~e...ha~ed
Fu~re
avail@k ~o ~,~sr~mam quh,e
C
~he~@), vec~>~
mm~mh~e hos~ ~mm~me res~x~se akI ~vhie%e kag.,~:s, h ~3>h:'~d ~veombi~'~a~tFe~e exp~sshm fOH(::¢i~}gdirec~ [:~ vho tr k~ ef ske/eta~ m~sde, ~ a4dithm, we ~ d to @vek~> ~o~e~ @ s fo~ the systemic delb:eu of mcombi ge~ses {<,>the I mu>¢:'k>mass e@ m~.}sde. Each of the~,e oh)Co,ires w.iH reasire m~£t ~.:,¢e h,~,,~,: ,scie~fific res>eavh i~ the aea:, of imma~~ek>g> viirolo~> a~d S~'~ expressiom F~@/y, it is es~,e~iat {o @sig~ cae{@~y a~d ¢arr) c,~at
A~ 8
Perhaps the most ~m:~r~isi~g app/icaior~ of m~sc/e-.hased ger~e has ~ ~e W e of' ~)~ym~c vacci~es, i~ v~ci~e es, i,m, i~j of piasmid DNA vectors ts in t ~ of a protei~ tha e:a~ serve as a~ amio ten m sli celt~hr i m m ~ e re i~ Ne i hmto Po|y~c v~cir~es are an ideal appl el" cur-rein musele-h N~e ~herapy ~ecN~Nues becaase: (1) tN goal of 171
~? W~#,'~~,~,A. ~ ~ ( t ~
L~4~
~ ~
1t!, t,l?f~,41t
I
I
~
I
I
I ,
& ' i ~ 11, [745-t 1!!
I
Ni~o I.L,, M I l L ML. i
llI
I,C~.d l l } I I A . , i
~.,~ ~'~. f ~
review, ~ ~ of ~ ~
Lh~~
~ ~ m~ as a target tiss~ ~or ~ n e t h e ~ , d ~ s ~ ~h~ that have ~ used for m u s c l e - b ~ ~ therapy, s u ~ ~ ~ di~se.~N~ using ~ and discuss some of ~ e obstacles th~ remalr~t~ route to muscle-rinsed human gone therapy, ~n~fia~ ta~eI tissue is e~e ef the cri~ica~iss~s 'd in desigNr~g a geee-theraW a ~ a c h for ~ y ~, tl-e choice of t ~ e t tissue is dictated by the he ~ a s e it.lL For e~ampte~ i~ is get,orally ay eNthelium mus~.N targe~ ~ g~ ~cessNt : fibre:sis. However, in m~y Nsea~s IN a~es o e ~ o r ~ N a s~cific cett tyN+ a+ we~ ~+safe~y criticN de~m~i~ants of the r~os~ iaN ; a ~umber of p~N~er~ies~hatmake i~~ attractive ~n ger~e ~herapy°First, broth ~eo~atN a ~ ~ult ily i&mifi~e and isolable s~emcell ~bx sketeta| myobl~.s ca~ N growr~ ~o I~'ge numbers i~ ~blytra,'~Nuc~ with rezombie~nt genes using a , including retreviru~s, liNfecfi~s and. DNAm~dified m inlo lll~fi~|.fatecJ d~ (i°~) ~njec~ier~ s~ch gene~icaliy myofibers te Ncorae stably i ~ sue. ~ N ~ k ~ m~ified ~ pr~e mrs g N~ins N vicm argdM vi~v, ~upp~yN skeleN m~cle Women,s the e~cien~ ~eim m ~ systermcci~cuIatienoNne~ly,~eNa| ~ne m ~ o w or va~ular e~dotheliN ~Ii.s,has a N N that heretically medifi~ mym~berscan. if rgicM|y m ~ v e d with,~utadverse com~equences. ~ t ~ i ~ d on ~ proposes ~ demons~ae that ~ y o b ~ can be u ~ b~Lhm produce musc|e ~ m s ~ y deliver physiologicallevels :rues and cytekines t(~ the systemic circulafief~ n m the Nv~ages of sketeta| myob|~s~s for | rau~le ~e rare ~ N t~ng ~press DNA ve,~rs fo~t~wNg dh'ect mpeny ha~ recently ~ exple~d m deve|~ that may ~ useful f~r a wide variety of iw us~ N addition m its obvious utility as a ~get erapy of iN,orbed my~aNies, ske~eN rausc|e
i
i~
7. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
a
Iegs, liea~ior~-defeetive
Ge~e~ic modificatio~-s
myobtast
Plasmid
M~s
~
,1~ I',
/ Myoblast hawest
e
b N~ob/ast
/
',,4,
il;
i.m. iniection
t
i.m,. iniecSi:on
iota. iniection
!: iI
Fusion
\~ I ! I
Skeletal m u ~ I e I
,
il
Unmodified mydibe~
Modified myofiber F~:~ 1oA ~hem~is ilusIra£io~0~Nf~ sIraI~icm {or m~acIeg~e lt'te~a~y.The sIeps i,~woIvedi~ the use ,o~(a} my®last transplantaSors it) ~epIcaIiom @(ective a@s/@dmta~4 (e} ~NA for m ~ - ~ d @r~sfsem~:yam @pic~o
is a~ excelent target {issae for geee therapy approaches designed to treal inherited aed acquired set,am protein def}c~encies and fbr genebased vacciees.
Ex vim and ie ~im ge~e ~herapy appr~aehes iarge~li~g skele~| mu~le of
~ e first m u ~ 1 or
a|ly
tN~r fled m
im;o|ved ~he h:m b ~ k into host ma~le
by i.m. i!ieclio~ (Fig. I: Table I)'~~:' ->:( In these sludies, normal cuII~red ~yobtast~ or 11~ .c~!:iqt t r a i l . ] i c e d i/l ~ifft)" w i t t replicalion-
defective re~mvir~s~',s or p=asmNs cX~+eSSir~gtie genes enc(ydieg I3galac~osidas~e, humae growth hormone or factor IX were reimplamed ieto sy~gN~eic mice by simple L,mo iriiection, By his~ehgical analysis, such/eeetically r~:x_tifi~ myobhs~s we~e shmvn to NEe wi~h ee@ygenoas ~ m s~ab~y ime ~he bos~ m ~ l e , i~ N the Lm. i!jection of mladvely se~ll eu s of such ge ly m / i t i e d myoblasts ~s~Red in Ihe ~ec of
167
my~Nast tranffer is a.n inhe:en~ly~ab~.>~oi~teasivearid e×{~:~sivepro cedric+ rwq~iringeither the is~>htionof pfma~~+Wob~a~s~Lrom cacti A t t l ~ h L ~ initial studi~ provide:l an important pr~.s~fof pfi+> tremied patent or imnveam+uppmssio~ ~bllowing m>nau{olegems myoNast woa~spka'*mtio~.Second, human m~'~e appear~ to ~ sign ci#e concerningL~ feasibilityof using ~ M and ge~edcaHy~ i + f ~ m y ~ h ~ Ns the gone therapy of ~ h inheritN myopath}esand aificmstly~essreceWiveto t~:~nsNa~tedmyoblaststha;e mnu~: ma+;c}e. Thm, re:+ + feces ~ s N myoNast tr~ms~er ~?~' to g~la~: ~ s i ~ mc~a~fut human g~rg therapy:.Fi~t+ cant a~t N ~ 6 × 1~ celi recent r e d s have NN myobhst N :~e~ts ~ y N e~anced by No-injection ir~j~ of fie gm~cle, dm~ fi~ngs remain + a~d it is ~ t ctear fffat they are app+ke&le m human therapff, h summon%akb~gh n~yoNas~~aasp~ was historicaJly irnv~am in d~elopi~g ma~te+~sed gcme therapies+ i~ m ~ ~ ~ matnxo Thin ~ n m mutat~ in is unlikely m play a major ro~e is hmm>ange~e therapy ~nless it is ~ssible m devek~ a uuiversN dc.n~rmy~L';}asttha~ca~ ~e efficier~tly engrafted into h~man muscle+or unless it is ~0ssiNe m develop tram+ ~ ~ (~ - A p~e~n ~morm invotv~din r~u{ming sieur immun~ppressive ~egimens that wrmit tor~g+termmy~{~t graft survival in huma~ mu~leZL - ~n~ contextof tN~ r ~ i ~ , the attacbmen~a~ e~tty Dimc~ DNA injection In ~ ) + ~\Nff ~ d co+w(wke~ made+the surprising th~ skete:~l can take up ~ d s~ab~yexpels im~ma~u~gdy ~ ~ ~ r~R ~ t~ m ~ ~ fe,~mgnDNAin~ a injec~M ptasmid DNA~, 'tNs finding r:d~d the ~ b i t i ~ y of uskg e~tL direct DNA injectio~ as a straightfem~vardmeth~ of M vivs gone - MMum+~ ~ + e ~pid~ t~at faei~iba~ the tranN~tN~rapy in m~ele. Direct DNA inj~'tion has a n~m~r of ohvir~s adwavmges as a gone tra~uction a~oach ( ~ b k ~}. F~rsL k is a simple in vivo gene transfer tectmique that does ~ requi~ ar~ iNi:cL~om~ ~ - ~ enzym~ that ~s p r ~ o m ~ y ~eal+z~ tkms vector m~di:hatis feud@ applicable m large s~m~m of ~es~ts Ptasmi&based vectors are easy to constrgct, a~d +he prepm~do~ and ~ of e ~ t ~ r proteins+ ¢ + # ~ m t e m ~ ~pi~,+ storage of large amusers of DNA is relatively swai + aMitiom the a~am~t of traasgene expressio~ is progerdo~al to the ~r~ ~ ~ ~ ~ain~g a r~r~ @pFK~phm~ re+tuber of DNA injegtions, and ~ a ~ e d injections c ~ ~ easily ad~thohym*mtegroup ~ d f~ta~es the t m n ~ t of tlmse protein~ mieisteredV2:( NnaRy, transgene e×pfessio~ aN:marsto be s~hle for perk~Js of at ~east 12.q9 momhs Nthd-,M~gdirect DNA iNe.ctio#:o - ~ stem ¢et~of m u ~ ti,,mm,ff is m~non~lear a ~ Despite these advamages, the usefialnessof' dir~xt DNA i~ectien has ~,~}~. It c~'~~ d~ffemnt~teto form new my~be~, or fuse been gready limited by the finding that the e~$ciescy ~:ffgone transfer folk~ing Lm. of pt~smid DNA g extre~rely lea*: ~ss Lha~ 1% of the myofibeesi:e the ~ of iv~'ecti~rtm~e up ~ d e × ~ s t ~ ptasmid DNAm DespRe ex~nsive vaxk by mmqyg~r~ps, it bus th~s *~ been m i~rease significantly~ of bg vivo gone ~aasfer fol|owmg Lm+~nject~ N DNAoH~'eve~; recur s~ad+ ins have s h ~ n that ~ |evets of t geae e x ~ s i o n l/oh lowing direct DNA injec~i~ cam be incma~.l at le~gst5(Lfo~&by i~cw~ing firm tran~ripfiona~ efficiency of the iffWaeaJ p~mid :?. Ti~e~ fin~ngs, N~g with ongoing studies designed m ~+v+,o}. mRNA stability and tm~sMional efficie~zy, a+ weii as to increase transgene or mI~NA c ~ numbers Mlewi~g direct DNA injegfiom T ~ - The +nlm~aO&mof fore~ ~ A ir~o mils in eel+ hold prmni~ for exparding the usefulness of this approach. Howevee, at ~ proses time, d i ~ t DNA i~jection ~ a r s m ~ u~fu! only -A ~ ~ h=.~b ~ ~tab~i + ~ intoanother for the development of D N A - b ~ vaccines that require lOwolevel ~ d wa~sient~ o m b i ~ t g~ne exp~ssion i~ n'm.~le (see below). T ~ ~ d ~ # - Th~ mr+ at wt0~g~a specific mRNA i~ Aden~wims r e c t o s R~qplicafiono~f~6ve ad~novini~s (RdAd) are one of ~e most promising vectors for in vivo gone d ~ a ~ in skele~ m~vAe+Adeno+ im eell~, an~ IN~mid~ are P/p~.BI viruses ~ douNe-swandedhuman DNA viruses that typically cause veer+ ~tf4imited res~xato~ tract ivdecfions+ RdAd in which ~ El ge~s have been replaced by a p~o~er-transgene eas~tte can effficiently #~ysio~%icd ~vels of ~ombiuam prmeins )9!9the systemic ci~u+
m
isthct ~(x~{ replicadsg ~d ~?o:~>~::plicair>g col types in v~v< b~£ ~ack the ability io geoOoe week T w o months em~e i@ec{~ous ~ g e ~ y . Tte abi}i~y ~0 isS:'c~ ~or>replicai~g cel types is ~'ticaAarly imo p,or~a~ fbr i~5~rive m~
8
Other vector zvsgemsofbrmus~@gone ¢kempy @her are as for m ~ t e gene l)o bare ~ = used m y in ~ a~ m~ for tN in rive of skeleN my of their iiVy m non skele.~l . i ~ ,
even
d
r~Jr,ises
y into
Di~a~ ~ fee ~ e b ~ o m u ~ J e o b a ~ d gene theraD' lar ies The m ~ obvie~as ef mu~ g.ene is ~ t of singlerecessive my , such as DMD~. DMD £ an X fnked my y tha ~fee{s ximaely 1 in ~as. ~a is by m of %e dys ge~, ~ i structural protein the has been 169
///
/
~
~mphm geac ~o +go e:dm+~'+e~y~a%+ee~+~e<'le
"
+Wa,ss Of (+*¢ h+a+'+a+o+~g/a+:++sm+++(}+~+s+++~+¢m~'}oP
¢ba+le~ge +~ rise. ge++e ff~ec@y of f~+g'~eri~+4 is ~hc @~eR+~em of a s~s+emi:,+:
++//
s~ei~k~ty
~mi~aJly ++~iaed myofi~rs i++ a m++veb+ + g e ~:+gam+++smsmeh am~rm+L
@
k l +m+m "..
&r+w+ p+vge#+ de~++:iencas A~+}~m~ ~+ mm+++a @m+¢+pi+msve+ +.at t++am~ab+echat+eliges +~J+ef+>~:++veger+e
++
f
@4Jaf+o~
a++++++++}++0 +MY++++{+~++'++++~-~+++'++++ ~m p+J+~+C+++g+
a ~eta~ve~y la,ge mi~@er of ~,eam ~ + @t++eie++¢+ a~+a~ ++ ~ we}| ~¢',,~ tO m++ag+.+.> ba+d gc~ +++g+u{+icstTabk+2}+¢+~+++ ~+++++'>+++ As @s++i~d +{~:+ve,myo+++i~+mca+ c a @ ge+m+~k+a~}ym~×fifieda++ cam efMde+~t+yse+ ~%m+.,
,
¢+Fe~¢
J~ + "-+... +%>_ +,++
+ TgPa++ ~g
+ + + +a~{++zmim tBempe+ti¢prc> te~m s+¢h a+ E++ m+,] f~++tO++ +X+ tJ+a~a+e ++X b~++a++, ++*em~i from myofi+vrs g)dr+ Pe+~ +++:+st++t+m+++emit c + +++:+,~t~m~+~a++++y+ m of a +e+mmpro+ m++~+eik+e~+:y is ++++h+im+a+~y+++¢h ++mp½r @+a*~ ++:~_.:+~+¢t{++>+~ of #+f+i++++eri~edmyopa~}~y ~ m + s e +~ig~+m++k' ~raa+~+te~i+meffMe+++ c++ am +~:o~reqm++J + o~+y a m+aivety sm++li
+',..
m++k ++eed+ ~:+~+ +++~+p+++temof +m~em+~ ++a+:++kvem~++:g~+m~++:e~++~c~ g ++++
~
~
++:e+.+~+ + + m
+iag+ i+m++r@cti~+++of R~Ag e+++c++i%E+~+
+++
@+ m
RPaA& T+P~ i+i++di~gsmg#,e~+~@m C~ Lm+ ~' RdAd h+++~+J~+ ? + + may ~ m~+ f~+ hu+~+m+ di++ad ~+i+s im+he +++ from+e+ A +r+~ Lm+ imp+m+ ~ +++~ of a p~+m+d+,b~d E+++e~+:~+s+ skin v+t~+ ~+ +a+ t++++++h+m++m +++it m kmg+~em~~ + s+v++t~+{s i~ m + (T~+~+ S.K+, Sver+~m, EC., BiaS+ ~.+ m+ ~+i@m+$M. LA h+a+rmm wo+#+ ++R~ait+ethe kin+ i++jeco i+~+
~ amJ t ~ +¢++ Lm,
eff+k~b:s of t+se p~a+m~ +++'+~+may dMv; t k +++ of m
i+++++++>+~ ~ m ~
~ ~+ t~++
~+e+ my~s~
~m ++f
+ ++ m+ +++f+ mi~ DMD+ Mos,
+a ~ m + Proem+
grea
+ / ~ Lm+ {R+RdAd a ~ d ~ { DNA ft~ ~ m ~ ~ y : i+~saes~ema~i++gm
~k~
' . . . . . . . . . . . . . . . . .
~
e, . . . . . . . .
,s,
~
.
.
.
.
.
ge~e e:,,~/'essk:< h@>v, i~~:~dee¢~ ,DXA
ex F~aHy, t
sio~ ~
a%' h
dm~ca~ ~als are,~.~derta%er~,.
hes
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
p>:~e m t2>
kaHy i~ tica~b:' ld be eva~ ir~
~%d <~i>~a~ ~
~s
@s'os
: s:ore~ge ~,;es hg~fhed ly I s~ div, ases (LSDs~ a'e a ~hkd class of di rs {ha may ['~:, ~qie~larIy well s~hed ~o m~.>c[e-di gr~e gqe . ]£-ese rare ~'~:cessivedi s a e e>:h ¢h~vc{erized h} ~he ah~em:v of a sisgle ~%~w{k~>~:}a~~ysesem;.~e~zyme ~ypicaHy rc:d~-. ir~g i~ the [ym~,m~ai acc~am@aio~ of the s~abs~ra{e ~a,u~o~'sor,,of dse mis@g or defhctive er~zyme, a@ @timae[y ~ea@qg {o ceHs/a dys.Nr~(:~ioe a~(i:or dea{ho C~£~icaH> @ese diseases presea wi~h mema~ re~ardadom f?om ~e~roea~ dysNm:'tio~:~,a~d e~a~,.#mest of the lh,er aed spleen, flora accommodationof g~ycosamineg~ycan s the£: orga>. Mo-s~ LSDs resah m m ehiid Them ~e c~rr-e~t[y so e~.ctire ~eatme~ts for aW N {he ~SDs, makhag eea: &sere the ap., higNy de@:aNe, The s sNl ther@y of LSDs wilt ~:~ost prot~hty ~q~,.~.h:e tha a Nzge ~age of cells i~ a ti {live< @eem eee{rJ em:~'o~s sy (CNS}] ~ceive the reph~cem-eg~ e~,zyme, ~xt~mae~y, h~eve~; ly a~ eraymes ~:~ssess a tageti~g sigsal,, ma~ g ae, ]~a~{s,cd/s ~avkir~ga ~¢:ific ~yso,,oma~e~:~zy~r'~eear~ u~a~ ap e~:~zymefrom {~:e >e,~mmvia their mamm.,:e 6-phosphate recep~@'s a'~d delg<~r this eezyme m the {y~:}mes~ {ieg the/ysomma/ deficiP~ <~kAs ~ h:~Fig A a viaNe thera[~:0~{ics~ y w~:m~dN h} mmN~x:e m~c[e cells wi~h @e ge~e e~c~ir~,g {he/y:,osomal eezyme of igteresL dh'ec{i~g {hose cells to pr~ace high ~evds of e~yme h~ ff~e serum, which co~d ther~ N taker~ ~@ by cells m the [[~ea ~;,pleee a~d CHS The hw:dks {o t~: overcome h~ the corrccfi@~ of ly~esoma~ ~omge by ~he are, [~ ~ru'~ csa~s, similar m de~: for k> the for ~z~{~m.o ies° Fksh Ngh s of ir~a~t expre>
sio~ N ~ d h~ m a c[imd , ~d, ~his e~ km m~st N mai~ai~d lbr ~he ~ife of the h'@ivldual, ly, amt m{~t i ly tbr di char~:~erizedby' , ~he r~" /ysosomag eezyme cross tN °~ai~ i~ m tN CNS lities of di ., less, tb~ ~gem ckmiN of the genes resgmo for most N Ne LSDs, aa~dthe avai|ability of N{h smalb aN Jarge-a~imd m~. e~s of the~ di~ea~s, shouM facilitae the deveh@o me~t N ~oelieiea/NNs tN/izi~g myob|ast lamrafio~and i.m. i~kc~io~, of RdAd or Nasmid vc~:tors.
ge~ ape es. ~I2~e f;easfbiH~y of ~si~g d
DNA i
as a v~cc
tie~ ~.'.r wi@ as exp>essk~q p}ee,m}d c
p% h~,u'
t < > , isch.@h~g he.rpe~, '.-
Fu~re
avail@k ~o ~,~sr~mam quh,e
C
~he~@), vec~>~
mm~mh~e hos~ ~mm~me res~x~se akI ~vhie%e kag.,~:s, h ~3>h:'~d ~veombi~'~a~tFe~e exp~sshm fOH(::¢i~}gdirec~ [:~ vho tr k~ ef ske/eta~ m~sde, ~ a4dithm, we ~ d to @vek~> ~o~e~ @ s fo~ the systemic delb:eu of mcombi ge~ses {<,>the I mu>¢:'k>mass e@ m~.}sde. Each of the~,e oh)Co,ires w.iH reasire m~£t ~.:,¢e h,~,,~,: ,scie~fific res>eavh i~ the aea:, of imma~~ek>g> viirolo~> a~d S~'~ expressiom F~@/y, it is es~,e~iat {o @sig~ cae{@~y a~d ¢arr) c,~at
A~ 8
Perhaps the most ~m:~r~isi~g app/icaior~ of m~sc/e-.hased ger~e has ~ ~e W e of' ~)~ym~c vacci~es, i~ v~ci~e es, i,m, i~j of piasmid DNA vectors ts in t ~ of a protei~ tha e:a~ serve as a~ amio ten m sli celt~hr i m m ~ e re i~ Ne i hmto Po|y~c v~cir~es are an ideal appl el" cur-rein musele-h N~e ~herapy ~ecN~Nues becaase: (1) tN goal of 171
~? W~#,'~~,~,A. ~ ~ ( t ~
L~4~
~ ~
1t!, t,l?f~,41t
I
I
~
I
I
I ,
& ' i ~ 11, [745-t 1!!
I
Ni~o I.L,, M I l L ML. i
llI
I,C~.d l l } I I A . , i
~.,~ ~'~. f ~