The potential of extrachromosomal replicating vectors for gene therapy

TECHNICAL

Focus

The potential of extrachromosomal replicating vectors for gene therapy

T h e need for improved vectors has emerged as the most pressing requirement for convening the potential of gene therapy into tangible results. Certainly new types of vectors would be valuable in simatiom where the target cells of interest for gene therapy are proliferat ng cells. Examples of such proliferating cell targets include undifferentiated stem cells, differentiated cells that retain the capacity for proliferation, neoplastic cells, +:q, and cells in the rapidly growing tissues of young patients p. CALOS (Table 1). These t.vlls may divide either frequently or M . t ~ rarely. [n either case, in order to keep pace with proliferating cells, a vector must either integrate into the Peeslste~,e OfDNA v~ors in target cells is advantageous chromosomes and, in this way, replicate along with file in mOStappffcations of gene therapy, Par~tcglarly w h ~ genome, or it must have the ability to replicate and he tat~et ceils are uadergoMg pro~feratlo~ rector Ioagevity retained extmchromommally. Even when cells are not wig depend oa ellh~ the t a t ~ l o a of the vector lato the dividing, DNA is rapidly lost from most cells unless it is chromosomes or the oblli(y of the veaor to replicate aad be relalaed extracbromosomaa); Vectors that efflcler~ integrated or has a mechanism for nuclear retention. The most widely used gene therapy vectors today tntegrate ie a nonreadom fasbloa are ¢mvcntly u ~ are based on retroviruses. Retroeiral vectors integrate and those that can repllcale extracheor, tommalty prot~de a major alternative str~egy. $etct~dasses of such into host chromosomes stably hy using a viral integrase. However, retroviml integration is random, creating a veaors are under developmea~ carrying mechanisms foe different insenional mutation in every target cell. This pgolonglgg DNA retent~ou ta marsmaNaa t o d d lb~t extead situation poses the risk of undesirable acth,atinn or vector I~fethee in aonproUferaling cells ns well The to inactivation of genes and also surrenders much of the vectors utl~.e either ebror~osomal or viral ~ s control of gene expression to context and position mediate r~lllcatlon and r ~ t e s ~ and have a large sLce calmcity for imertfoa of genes of interest I discuss Ihe effects mediated by the random target sitel,2. Strategies to hring about the covalent integration of a gene of state of the art for these v~ors, itw~dlng the assets awl interest ir.to a host chromosome in a site-specific man- limitations of theiPf~ture use ia grae tberap3'. ner are attractive in principle, bet have not t'~en acifieved in practice 5. Many of the other systems being tested for use in autonomous replit+ation in mammalian cells6,7 indicate gene therapy, such as vectors I~sed on adennviRts, ffiat sequences that can mediate replication initiation adeno-associated virus and herpes simplex virus, can are frequent in human DNA, so it is likely that no speneither integrate efficiently nor replicate extrachromodal provisions for replication are necessary. To mainsomally 1,2. Therefore, survival of soch vectors in prolif- tain a linear conformation, the chromosonle ends must erating cells is limited. Simple plasmid DNA vectors lack be capped with human telomeres. T h e ~ telomeres are DNA sequences that enahle them to be efficiendy repli- composed of the repeating hexamer TTAGGG (Ref. 8), cated and retained in mammalian cells'i. Thus, cun'ent and it has heen shown that this seq~.lence is sufficient to mediare fomtation of a functional telomere 9-t2. vectors do not provide a completely satisfactory" means of being propagated in proliferating cells. However, a series of vectors tinder development have the abilily TaLE 1. Gen¢ therapy appEcations for proliferating to replicate and be retained in the nuclei of matrarmlisn cells, These vecPmslble therapeutic me tom use either chromosomal or viral cdltyt~ l~t.amples sequences to achieve replication stem cells Adenosine deamin~ Hemstopoietlc and retention (Fig. 1 and Tahle 2). deficiency, sickle tell Delivery of the vectors by non-viral anemia means will avoid most of the safety concerns associated with viral vectors. Differentiated cells V'ascularendothelium Angiogeaesis in tumors with proliferative AIDS, cancer Artif~ial chromosomes capacity T *.'ells immunolhempy A theoretically desirahle vector for gene therapy would be one whoUy Hepatocytes Hemophilia composed of native manlntalian dwomosomal components, includBlood vessels al~er Restenosis ing origins of replication, telomeres Regenerating cells surgery and a centromere5. How |hr are we from generating such a molecule and Neoplastic cells Tumors Melanoma what limitations would it impose? A primary requirement of an artificial Growing tissues in Cystic fibrosis Devdoping lung chromosome is that it should have earIy life epitheltinm in infants the ability to replic2te. Sindies with TIG NOVI-:i~tBER1996 VoL 12 NO, 11 fxlpl'figtfl© 19~1EI~ t~ ,~itmc¢ Lid.MLrighLs~ t ' d

pII:501(h'L9525~gfiblt~Mg.X

u 1{~4tl5259f+$15 t~)

463

TECHNICAL

FOCUS

(16.2kb) mediate efficient replication, but do not mediate segregation t8,19. Because of the difficulty Tetomeee or/ Centromere of cloning large expanses of centromefic DNA, it has not yet been F///dAI I ......... . . . . . . . . ' ......... possible to reconstruct replication, telomete and centromere elements T~lomer0 into a functional artificial chromoCentromere Gene of interest iT////~t some in mammalian cells. I I I A more productive approach has been tile progressive reduction of existing chromosomes using telomere-mediated fragmentation. in this method, imroduced frag(b) (c) env famy ments bearing telomeric DNA inteol repeats grate into chance chromosome EBV o~iP Human or/ breaks, removing the natural end of the chromosome and fomling a new end 9-t2. For example, repealed cycles of such a protcmol Bm~ d have led to a hutmn X chromointamm some reduced to just 8Mb, i ~ m a EBV starting size of 164Mb (ReC 12). ~gN.4This chromosome pos.se~,es about 2Mb of centromeric sequence, two tdomeres, and re#ication activity. Gone of tnterest It form.~ a minichromomme wiffi stability comparable to that of Ftc~trl~1. Extrachromosomal replic:ttlng vedl:ts, h) A protoQ.~ of an artificial full-sized chromosomes. Additional chronlo,~onltl vt'ctor. CenlmnlelScst#qnelxtxL~are pmporti,m:dly much I:lrger thin shown eyries of telomere-mediated fraght,rc. (b) A vector Imst'd tm EItVctnnlxments, oriPconsi:;ts of tile family of repe:a mentation m,ly succeed in further elements and a dyad symnletrv element. Both dements along with I;2JN:I-I are lequired reducing the size of the centroftJr replicatiot~,in addition, the f,lmily t~f rcl~+ats and P:B:X'A-1are required for nuclear retenti+m. (c) A vector ¢ith hunlan sequences to medi:ae replication and the EIiVfamily medc element necessat 3, to stably of repeats and IZ'CtI-I sequences ft}r nucleal retention Element,~v;ith similar fundions nlaint~in the minichromosome. It is alr~.'depiett~ with the same sh:lding: bl~lck, re#cation; gray, nuclear rdention; sttlped, likely that some minimal size will teitmlen:s; whitt', the gent of interest fclr gone ther.lpy. For zizes, :-,t.'efable 2. I ~ found for the artificial cfironlo~ m e tfiat c~nnnot I ~ reclu~.x:d further without significantly interferMore dM'icuh to ammge is the ceotromere Mam- ing with chromosomal stability. The final size will malian centromeres appear to have ~1 minimum size of probably I x driven prirn~Jrily by tile minimal size of ix at least s e ~ m l hundred kilobases (Refs 13-15). In stable cent,"omere, which appears to be about 1Mb human cells, tilt+ bulk of tile certtrotneric DNA is coln- (Rd. 20). pa'.;ed of tandem rep~.ns of a 17I hp sequence known A vector nf this size is f;tr bt.~'ond the size of vectnrs as :llphoid DNA {gel. 15). ExpatL,ies of al#1oid I)NA, in current use top gene therapy and poses problems of when transfec'ted into nlammalian cells and integrated major dimensions, p:trtieulady for tile manufactnre and randomly into the chronaosomes, exhibit some ~.~atures delivul 3' of vector I)NA. Therefore, while constmctinn of centromeres, m~gesting that ~llphoid DNA is of artificial chromosome vectors has not yet been realinw~lved in cenlronlere function It'.IT, Whether any nonized, once it ts, a seri~,s of challenging technical harriers al#1oid .~qtaertces are normally involved in ,:entmmere will have to be snnrlounted be~'ore such molecules fum'tion is not yet ,:lear. SmaU pieces of alpboid I)NA cutthl reasonably be nmd us gene therapy vectors. (a)

T.~,mn 2, Replicatit~',~efors Veoto¢

Replication conqlonettt

Rettntlott comilomrtt

Base vector sLzes

Patative host range

Artificial chromosome P,untan genomic DNA

Iiuman ccr:romere

1 to several bib

MI rrianlmalian cells

EBV vector

EBV or~Pand EBNa-I

EBV family of repel!is and F.BNA.1

6 kb

Primate and dog cells

Human ortvector

Human genomic DNA

EBV family of relx'aL~

22 kb

All nxaamlalian cells

and EBtVA-I

aWithout gene of interest for gene therapy,'. TIG NnVEMBEK1996 VOL, lZ NO, l ! 464

TECHNICAL

FOCUS

F.BV-ba.~I vcctom Given the large size of artificial chromosome vectors and the challenges of working with and delivering them, it has made sense to pursue simpler vectors that preserve some of the favorable features of artificial chromosomes. Ahematives to native chromosomal sequences to mediate replication and retention of the vector can be found in the human herpes vires Epstein-Ban' virus (EBV). EBV has a cis~acting origin of replication that occupies just 1-2kb (Ref. 21), compared with the 10-20kb required to obtain efficient autonomous replicatinn using chromosomal soquenc~'s~,7,19. TI~ rra~-actlng gene product of the viral EBNA-1 gene is required to activate the viral origin and occupies an additional 2 kb (Ref. 21). The EBV origin of replk:ation limits replication to once per cell cycle under correct cellular control, an important attribute of a stable vector ts.~. A further reduction in vector size can be obtained by using EBV's nuclear retention mechanism in place of a centromere. Part of the EBV origin of replication, the family of repeats, in coniunctinn with EBNA-1, which binds tightly to the repeals, is sufficient to prolong the retention of linked DNA in tile nucleus of mammalian cell#. This retention mechanism probably involves a noncovalem association of the EBNA-vector complex with :lie chromosomal scaffold. While this interaction can prolong the retention of DNA in the nucleus, it provides random rather than equal segregation of the vectors to daughter cells upon cell division, Furthermore, the stability of the vectors is lower than that of native chromosomes. In the absence of selection, vector DNA gradually disappears from rapidly dividing cells over a period of approximarely two months 23.-~1, EBV-hased vectors car W the EBV origin of replication oriP and the EBNA-1 gene, along with the necessary prokaryotic sequences for propagation of vector DNA in Escbetgcbia coli, This arrangemem produces a compact base vector size of approximately 6kb, to which a gene of interest can tm added (Table 2). For example, the mility of this type of vector carrying the CFTR gene, defective in cystic fibrosis, has been demonsttattd, with tile vector providing gtme expression for at least two month.'; in transformed dividing human airway epithelial tells in culture z~. EBV vectors can carry very large fragments of genomic DNA, up to several hundred kilobases (Refs 25, 26), giving these vectors the capacity to can3, multiple genes or native versioos of genes complete v
the continued division of target cells for a prolonged period of time. Unfortunately, use of a viral origin of replication also poses some disadvantages in proliferating cells. Like most viruses, EBV has a specific host mr~ge. EBV vectora have been shown to replicate in a variety of human and monkey cells and a dog cell line 28. ltowever, the vectors did not replicate in any of several rodent cell llnes~, 29, eliminating the possibility of testing them in rodent model systems that require vector replicaiion. While EBV vectors have been shown to be stable to rearrangements and other mutations in a human ceil line30, their stability and ability to function must be tested in a wide range of cell types to determine their potential utility. For example, it has not yet been demoostmted that EBV vectors replicate and are retained in primary cells and in animals, as opposed to the immortalized tissue culture cells where all the work has been done to date. The EBV system requires the EBNA-1 protein for replication and retention. As a foreign vital protein, it is theoretically possible that EBNA-1 will ~ause an iwanune response or a toxic effect. However, EBNA-I is not an oncoge,v: and is notably non-immunogenic 3t. It has been suggested that the properties of EBNA-1 that render it invisible to the cellular immune system might also protect the target cell from an inunune response elicited by tile imroduced gone of interest a4`

Chromosomal or~ vectors To remove rome of the limitations of using a viral origin to provide vector replication, a class of vectors has been developed that relies on native human genomic sequences to mediate vector replication, Recognizing that the EBV family of repeats and EBNA-1 could provide nuclear retention 6, we used these ~quences to screen a library, of buman genomic sequences to isolate those that could mediate replication in human cells. This study produced a collection of fragments that could mediate el~qcient replicminn whert introduced into human cells6. Vectors carrying such fragments were found to replicate with a timing and efficiency sinlilar to those of chromosomal DNA and had tile controlled, once per cell-cycle behavior required for stability of an extrachromosoma] replit~ating vector6,7.18,19,2~).32~ Vectors based on human origins give rise to expressinn of a gene of interest cloned into tlle vector for approximately two momhs in rapidly dividing cells, As expected, vector longevity is related to the frequency of cell division. This effect was demonstrated ha human 293 kidney cells, as well as in human lung epithelial cells and in rodent BHK cells~. Unlike the EBV origin, ffiese human fntgments do replicate in rodent cellsag, prot'Jably hecause all mammalian cells share the same sequence requirenmnts for autonomous replication. Therefore, it is likely that these vectors will replic:ate in a wide varieD' of malmnalian cells, using tile host replication apparatus. For ~ o n o m y of size, these vectors use file nucl~tr retention mechanism of EE,V, This nuclear retention hTstem appears to function independently of replication and also provides a significam advantage in slowly dividing and nondividing cellsL in vectors using genomic sequences to mediate replication, the role of EBNA-1 aplX~ars to he conl~ned to nuclear retention of vector DNA,

TIG NOVF.MBER1996 VOL. 12 No. 11

465

TECHNICAL

Then:fore, it is possible that alternative viral or celkflar sequences coukl be found that mediate nuclear retention with no requirement for any viral proteins. As with EBV ",lectors, tile replication and retention of human or/ vectors still needs to be demomtmted in primar}, cells and in animals,

Conclusions Table 2 summarizes the properties of what. at present, appear to be tlle must promising extrachromosomal replk~ating vectors. Artificial chromnsomes represent the ultimate vectors in the sense of retaining tile full stability of authentic chromosomes, as conferred by the centromere. However, the inclusion of a fully lunctional centromere is likely, to render artificial chromoseines at least 1 Mh in size. This large size makes all aspects of construction, manut~cture and delivuiy far more difficult, compared with smaller vectors. "vectors Ixlsed on EBV can provide replication and a significant amount of stability, while sLfll retaining :! small size that is easier to utlapt for manufacture and delivery. However, EBV vectors have limitations including dependence on a viRll protein and a limited host range for replication. A middle ground is occupied by vectors that replicate with dlrolntx~lmal sequences, but use EBV sequences for nudear retention, The.se vectors appear to replicate in all mammalian cells. While all these extrachromosomal vectors kick a mechanists for integnition into tile genon'4e, some random integration events are probably unavoidable. However, th W are rare and ;Ire unlikely lu pose a significant problem. Because of their mlclear retention properties, all el" these vector systems should also be advamageous in nondividing cdis. where they will increase vect{lr longevity. If delivery methtxls are effective and non-thrash,e, sudl as intravenous injection or aerosol delivery nf vector DNA cnmplexed to cationic lipkls, then repeated deliver}, of easily manufilctured vectors at intervals of a month or two might reake sense. Such a prlxoct}l might Ix: more appealing than less frequelli ddi~x.ry of at Iltort' stable vector that is, however, ovugwhelnliilgly difficult to work with. It bears remembering that penlmnentxa is neither required nor desirable in every situatkm. Vectors thai :ire gnldually lost over Iitne carry a huilt in safety feature against unfi)reseen adverse conseqnences and may be more acceptable to patients. Pernlanence is als(:, moot when tile target cells dremseh'es have a limited lifetime due to tissue tnrno'¢er, l)esttaction of tumor cells is an example ,.:'here kmg-teml vector persistence can be tmneces,~r}', Each gene therapy application l'tlns[ J~ evaluated for tile nlnst desirable level nf vector pemlanence. For applicatkms in which alxsolute permanence of tile vector DNA is desirafile, tile ultimate choice is bee,vcen exlrachmmo!;tmlal vectors with stability comparable to tile dlromosomes, and vector DNA atrsining permanence through covalent linkage to an existing dlronlosome. If non-viral vectors that can integrate specifically can be devdnped, they might be viable altemati'¢es to an artificial chromosumtr. At present, neither artificial chromosome vectors nor efficient non-vital integrating vectors are available. It will take much work to nlake these theoretical ptxs.sibilities into workable gene therapy strategies, but they represent worthwhile goals.

FOCUS Acknowledgements 1 thank Kevin :qawuika ful dtawh'l~¢Fig. 1 and th..: Cystic Fibrosis Ftmndation and the American Cancer Society for support.

References I 2 3 4

Mulligan. R.C. (1993) Science260, 926-932 Miller, AD. 11992) Nature357, 455-460 Vega, M.A. (1991) Hum, Genel. 87, 245-253 Wohlgemuth, J,G. et aL (1~361 Getle 7"bet; 3, 503~512 5 Huxley, C. (19-)4) Getle Tber. 1, 7-12 6 Ksysan. P.J., Haase, S.B. and Calc~s,M,P. (19891MoL Cell. Blol.9. 1026-1033 7 Heinzd, S.S., leaysan, P$., Tran, C.T. and Calos, M,P. 11991) 31ol. Cell. Biol. 11, 2263-2271 8 Moyzis, R.K. et aLt 1988) Prec. Nail. Acad, Sci. 6~ S. A. 85, 0622~o6~ 9 Hanish, J.P.. Yanowitz, J.L and de lange, "L ( 1994i Png'. ~Vatl.Acad. Sci, L~ $. A. 91, 8861--8865 10 Barnett, MA. elal. 11993) NucleicAcldsRes. 21, 27-36 11 Fall, C., Fantcs.J, Gtxldfelh~w, P. and Cooke, H. (1991) prec. Nat/. Acrid. SOl. L~S. A, 88, 70116-7010 I.~. Fall, CJ. viol. (1995) F-IIBOJ. 14, 5444-5454 13 Tyler-Smilll, C, e! aL 11995) Nat. Genet. 5, 368-375 14 Itmwn, K.E. el al. 11994) Hum. Mid. Genet. 3, 1227-1237 15 Wiltard, H.F. (1990) Trends Genet. 6, 410-416 16 I.arin, Z,. Flicker, M.D. and "l~ler-Snlilh, C. 11994) ltlfoI ,tloI. Geller. 3. 689-695 17 Half, T.. Warburton, P,E. and Wlllard, H.F, 119-)2) Celt70, 681-696 18 Ilaase. S.B. and C.alos, M.P. 119-)1)Nucleic AcMs Res. 19, 5053-5058 19 K~'san. P.J., Smith. J.G. and Cares, M.P. 11993) Mot. CeU. Biol. 13, 2(~8- 26t.~ 20 Karpen, GH.. Le, M-If. ars.l 1,2. H. (19961 St/elite273, 118-122 21 Yale:;,J., Warren, N.. Reisman. D. and Sugden, l:k 11984) PlOt, Not/elCllCJ. Sci, IL S A, 81, 38116-3810 22 Yates, J,L and Guan, N. (1991).L t'lml. 65. 483-,i88 23 D..i, I).C et aL { 199fit Gene 1bet. 3, 427-436 24 Ikxnetiec, S., l,ivant)s, E. and Vos, .I-IVl.ll (1~-)5) N~II Med. 1, 1303-13t)8 25 Sun, T-Q,. Fenstunnacher, D A and Vos, J-M,It. 11994) ,'i'~tl. Gt'~lcq.8, 33--41 2 6 Slmpmn. K.. McGuigan. A. and bhtxh'y, f" (!90fit MOL c'vll. Biol lf. 5117-5126 2~" Gate, X. alld 1!u:mj~, L. t 199e,) Getle Tber. 2. 710-722 2 8 Y;aes, J,I.,. \Vart~en.N. and Sugden, B. 11985) Natuw 313, 812-815 29 K~'san, Pd. and Cake. M.0, (19-)3J G'ene 136, 137-1,i3 3 0 Dultridge. R B el al. (lt~7) Moil Cell. Biol, 7, 379-387 31 Klein, c; (10941 61dl77, 791-793 3 2 Krysan, P.J. and Calos, MP. (19911 Mot. Cell. BioL It. 1464-1472 I M.P. tALes ( c a b s @ b a a n a t s t a n f o r ~ e d u ) ts IN n m } ] Dneal~rMEmro~ GE.~C.~ Brlolt~o~o U~IWRSlrV $CUOOLOF i MZ~sCIN~, SraNt'ol~ CA 94305, USA

TIG NOVEMBER1996 VOL. 12 No. I t 466

]