CA2202664A1 - Preparation of rep-negative aav mutants and cells which can be used therefor - Google Patents
Preparation of rep-negative aav mutants and cells which can be used thereforInfo
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- CA2202664A1 CA2202664A1 CA 2202664 CA2202664A CA2202664A1 CA 2202664 A1 CA2202664 A1 CA 2202664A1 CA 2202664 CA2202664 CA 2202664 CA 2202664 A CA2202664 A CA 2202664A CA 2202664 A1 CA2202664 A1 CA 2202664A1
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- Prior art keywords
- rep
- aav
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
- C12N2750/00011—Details
- C12N2750/14011—Parvoviridae
- C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
- C12N2750/14141—Use of virus, viral particle or viral elements as a vector
- C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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Abstract
The invention concerns the preparation of rep-negative AAV mutants and cells which can be used therefor. The invention also concerns an expression plasmid used to prepare the cells.
Description
IN 111~ CANADL~N PATENT OFFICE
In The Matter of Canadian Patent ~rp1ir~1ti(1n-Applicant : Deutsches Kr~f~ ' ~ Stiftung Des o~r. Rechts; et al PCT Serial NO. : PCT/DE95/01429 Fihng Date : October 12, 1995 Title : I`~. ~ Of R.,~ dti.~ AAV Mutdnts And Cells Which Can Be Used Therefor Agent : Ivan St~ 'y Our File : 08-875722CA
Date : Apdl 14, 1997 The C, of Patents Ottawa-3Iull, Canada KlA 0C9 Madam:
NOTICE UNDER RULE 160(4) Until either a patent has been issued on the basis of the application or the apphcation is refused, or is otherwise abandoned and no longer subject to t, or is withdrawn the ~l is requested to lunit any: for furnishing a sample of the deposited material to an ' I l expert nominated by the C, in accoldance with Rule 165.
R~irertfil11y submitted, GOWLING, STRATHY & H~NDERSON, Agents for Apphcant.
Gowlin~, Strathy & Henderson lZ . f I rK . l9g ~ g HUBER ~A O 2 2 0 2 6 6 4 ï 9 9 7 0 4 1 4 NK . g3~ 5 . c /11 . 1 Pr~visics! o~ rep-~eSati~ ~AV ~utl-~t3 a3d cell3 w~ch can be used t~ere~
~he pregent invention relates to t~e prDVisioD. o~ . rep-negative AAV mutant~i and cell3 whJ ch can ke used there~or .
rAurthermor~, this invention ~oncerns an expressiQ~ plasmid u~ble for the prep~r~tiDn o~ the cells.
Ad6no-as3Dciated viru5es (AA~s) are r ing' e-strauded D~A
viruse~; hel nnri ~ to t~e ~amily o~ parvoviruses . AAVs ~e*d ~elpe~ TirUSes~ pa~ticularly ade~oviruses or herpesviruses/
~or the replicatlon therso~. In t~e absenoe of he~per viruses AAVs iLterrate into the host ~ell genome, particula~ly at a speci~ic s ' te D~ cn.romDson e lg and 17, respectively, Three viral run~tions wer~ rAl; ~ q on th* 4 . ~5~ ong, l~rLear geno~e or hurnan AAV-ty~e ~. ~he ~45 bp lcrg "ir.verted repeat=A~! serve as r~r1i~A~ n origi~ and as ois ~ignals ~or iut*gratiQn a~d packaging. Ihe c~;.a gene cDdes ~or three structural protein~ and the rep gene for a ~amily o~ lti~ nr~ihn~l regulator p~oteins. Iqle mRNAs for Rep 78 ~nd its C terrrl~al-sp~iced version Re~ ~B start at ~he p5 p:~onLoter Two ~ terminel t u3cated versions o~ Rep ~7~
and ~*~? 68, llantc'y Rep 5;~ an~ Rep 40, respec~ively. are expreAsed under the cortral o~ thA pl9 prornot*r. R*p DrOteins ~re n*c*ssary ~o~ th* ~NA r*Flication o~ AAV. m addition, they are ra~Iuirad ~ar the oerle reoulatiDn a~ AAV.
AAVs suppress the tu~or ~evelooment in animal5 Fur~he~no~e, they suporess ~he c*l~ tranS~OrmatiQn aaused ~y onGOge~es as well as the induced r)NA ampli~icati4r. In addltion, AAVs have a~ anti-proli~erati~-e e~ec-iveness.
Re~ proteins o~ AAV are held r.DArpf~ e f or the a~ove acti~iti*s. ~owe~rer, an ~qg~ Anr o~ these activities to th~ indi~itiual Rep proteins and domai~s ther*o~, respe~tively, do~s not exist. ~ut ~ch ~n a~sign~nen~ would~--i .
-lZ. ~PR . l9~ 01a HUBEFCA 0 2 2 0 2 6 6 4 1 9 9 7 - 0 4 1 4 NR . 9 30 5 . 3/11 - 2 - !
.
be r~ecessarv to ~e able to th~r~re~tic~ ly use ~A~7s. A
r~s; h~ y o achie~ ng this aasignment consists in the investi~ation o~ s h~vins mutations in the Rep protQins.
Many experi~:lents ~av~ been n~ade in thia respecc. ~o~ever, the provision o~ rep-r,egative ~ tarts wnich are f,rse~.
~rom wil d-tyoe .~AV, has not succesded S4 far. B-~lt tbe~ are o~ligatory ~or the above investigation~, There~ore, it is ~}~e object o~ the present invention to provide a mean6 by which rep-ne~ative AAtt mutants can be obtained withcut the ~ove d~ baake.
Ac~or~ing to ehe inv~ntior. this is ach~eved by ~he ~ubject tnatters defi~ed in the clai~
I'hus, ~he s~aj ect ~na~ter o_ the invention ~elates to cells which staoiy express the A~.V Rep Protei~s 7a and 52 s~ ~ell as 40 and/or 68. Cells ~hich ~xpress the ~ep proteins 78, 52 ar.~ ~ are pre~errec.
Cells accor~ing .o the invention car. he prepared as usual.
Cells o_ the ~c~own lire ~;eMl are avarably use~ as iriti~l mater~al ~c, ''Sth ~vc~rirl~ Workshcp", Ch~ystal Riv~, ~lorlda, U.5.A., ~ovember 10-14, 19g3)~ Thege cells ~p~ess th~ 7 R~p proteina ~8 ard ~2, ~he Re~p 78 e;~?rYsaion beirlg controlled by dexamethasone- i~ducibls ~Tr~-L~rR Cellg of ~e~?~ are tran~ected ~ith an expx~ssion plasmid ~odir.g _or Rep 4~ and/or one c4ding ~c~ Rep 68 and an expresaior~
plag~id whic:s ~odeg _or both ~p proteins, respectively.
exp~esaicn plasmid for Rsp ~0 is used pre~erably, the expressio~ pla~mid pCMRep 4C bei~ r~c;~lly preferre~. Tt contair~s ehe D~A coding ~or ~ep 4~ ~et~oeen restrictior.
sites ~rotl ar~ X~al c~ the ~nown ~ector pX~X-2-XI ~
Rit~ne~ . et al., ~5ethod~ ~ol. C~ Biol. 2 ~l991), 176-181). pCMRep 4~ was ~eposited wit~ the I~SM ~Ce~mar. Type t~oll~ctio~ o~ Mic r 4o ~ isn~s and ~ell C!uleurea Gmb~) tmder DSM 9491 on Oct. 7, l9g4 and under DS~ 9488 on Oct l9, 1Z.~PR.1g~7 12-0EI HUB~A 02202664 1997 04 14 NR gs0 5.4i~l 1 l9g~. It also oelorhgs to the s~iect matter o~ tk~
in~s~tioIl.
Tke cells Gbtained ~3y mea~s or LLCI~ LiQn o~ pCMRep ~4 stably expres6 the AAV ~ep pr4teins Rep 78, Re~ 5Z ancr Rep 40 These cells -~ers deposited as ~ell line ~e 10-1, Ee ~2-2 and E~e 5-5 with che DS~q u~der ~SM ACC2193, DSM AG~2192 and I~S~q ACC2131, respe~ti~rely, or. Sep. :~&, 1994 Furthermore, the cellY ~ere d~posited as :ell lire KeCMlg with the DSM under ~Sgl ACC21aS on A~g 3~, 19g4 The a~ove cells also ~e'ong to th6 a 6ubje~t matter oi~ the irven~ior Another s~7ect IDatter o~ ~he irvention relates to a pro~e6s ~C~ pro~riding re~?-nesative ~AV mutant6 Such a ~roces6 ~omprise6 the i~ollcwing proces~ing tep~:
(a~ trar.s~ection o~ ceil ~ rr~7:n~ to the inver.~ion ~7it~
the DNA c~ a rep-nègative AAv ~utant, ~! treatme~.c c t~e ~a~s~ec~ed cells o~ (a~ with a mea~7 eraoling an AAv replicatiou, p~rti~7ula_1y an A~l~-helper ~ irus, a~d (c) isolatio~ Qr ~he rep-regative AAV mucants obtai~e~
Ib) ln ~ pre~erred ' ~ , ca-tr~s~ectior with Ar expression 7~7asmid coding for a glucocorticoid reoeotor takes place il procesEir~ step ~a) . A p~rson ~killed in the a~t is ~ami' iar ;herewit~ ~or example, he kn~ws the e~YpresEicn plasmid HG0 (c~. Rumar, V, et al, Cell 51 (19E'7~, 941-9~1) Fnrther~ore, proCessing step ~a) il~lplieS tEle in~e~ion o~
oells a~:ordi~g to the irLventior. ~vith a ~ep-neg~ti~e AAV
mutant _n addition, the person ski7 led in the art iE
~arn~iar w~th all t~q~.nique~7 ne~es~7ary to carry ont the a~ove proces~ing steps. Re~ererce is made ~o MaDiati~ et ~1" hlol~.11r~r Cloning: A la}~oratory manlla' ~19~2), Co_d Sp~i~g H~rl~or, ~7eu YQrk, 3~y way 0~ Sl~rrlPmP~ ~
':, _ I
lZ.~lPR. 1397 12: E11 HU~EFCA 0 2 2 0 2 6 6 4 1 9 97 - 0 4 - 1 4 NR. 930 5. 5/1~
, . ' I
_4- !
The ex~aressior~ NA of a rep-negative A~V mutant" cosprises an ~ genome Pptionally present in a vector and having n~ in t~l6 rep ger~e. Such ~ nn~ r~ay be partiaularly ~eleeiona, ~ns~:~tion6 and~or substi~utions o~
one or more nucleotides. Tb.e AAv-r~A mqy als4 'oe a de~6tion o, the e~tire region ~oding for Rep. ~n ad~ition, the l~:~A
coding for Rep rita~r ~cartially or ~ully 4e replaced by a DNA
coding +or a ~oreigr protein ~ arei~ ~eptide) G~d ~y a DNA
coding for an ~n~; ~Pn~ ~A, ~es4ectively. The ~oreign ~rotein (+~oreign peptide~ aIld the I~Rn~; ~Pn~e~ll ~'~A, res~e~tively, ia prerera~'oly suita~le ~or gene-tke~apeutic asu~es. ~husl ~he ~co_essian ~rep-negative ~V mutant also i~Lplies tke ~err. "rep-negG~ive A~V vector~.
M.oreover, the expres6ion "DN}~ o~ a rep-ne~ative A~V T~tant"
~lso camprises a ~NA ~hich in ~ddition to the aoove-Tn~ =d ~s~tR~;~n~: includeg further mu~ation6 in otker AAV~ A regions . These may be e . g . t qt; ~ln~ iTl the cap oene. ~n ~uch ~ ca9e, ~ t is re~uired ~hat an P~rc~ hl P
P.AV cap ~ere ia prese~t in tAe cell3 ac40rdirg to ,,~e inveTlt~ on. It m;~y be in6erted by means enabling the AAV
rep~ioation, e.g. the AP.V helper TJiru~. The person skill6d i~ the art i~ familia~ with methods OT- inserting a ~Av cap gene, e . g . in an ~V helper virus .
~he expression "A~V heiper vir-~6~ compri6es viru,es which en~ble a replication o~ AAVS, Thes6 viruse6 l_e pa~ti4~1~rly ad6no~rirus~ sucn as adeno~ u~-z ard he~p~svi~uses .
13y means o+T the present in~rention it is possi'ole to prepare ~ep-s3sga~ve AA~ ItL~ta~ts. ~hey are fr~ f~om wi:Ld-c~p6 AAv, since ~ ' 'Tl~t~n OC~lT~rpn~c as h4rpf-T~ ir, t~e t-;~Tl~ e~pression of ~ep pr4teinE in c~lls, are avoided.
Thu~, the pres~IIt in~r~ntio~ ~ep~egent6 the basis ior restricting the acti~rit~ e~ ~s~ri~sPd to the ~ep proceins ta indi~idual Rep proteins and don~irs thereo+, resp6ctively.
I
lZ. I~PR. 19~77 lr: 01 HUBEI~A O 2 2 0 2 6 6 4 19 9 7 ~ 0 4 14 NR . 930 5. 6/11 -- .7 --Th~ s makes possible tb ~nvestigate in ~etail the mode o~
actio~ of A2.7r as tumor-sup~ressi7~-e r7~in~7r~ hich is ;nrliqr-,nq~hl-~ ior t~e use oi AAV in t~e t_eatment o$
tumo~;. Fu3:~he~o~e, ehe preserc invqr.tion o~ers.
~?ossihilitv of erovidin~ rs~n~h; ~r~ ~vs which qa~ be us=o as viral vectors ~o~ cene therapy. ~ep-~e~ative ~æv mtltants Ar~r~rr~;nn to the invention :r~ay bear ~enes ar~ gere s2ctions, respectively, usable ~or thiA p~rpose, ~ney ~ay be locatec~. ~articularly in the -ep gene an~cr cap ~;tene.
Thue, the presert inver~tion represent a break-through i~or the p7-r~r~rr~; nr of veceo~s usahle for ce~e cherapies.
_~ ~rie~ de 7cripeio~ c the dra~lrl~:
Fig. 1 shows a diagram o~ the Rep-cocillg DNæ in tke pC~ep40 expressior. p zs~.id. The start-.~l'G tri~le~t an~ the t~7~A~;nn codon ~,A are ircicated, ~hey co~re~pond ~o those oi t~e ~oild-t~pe-AaV ger.ome. The intron ~posieior.:
190~-2227~ is removed ~cy site-directed ~nutagenesis, so chat ~eo ~0 can be expressed witho~t splici~g.
The ir,vention is r~Ynl ~ i n~ s by the examples .
E~ample ~- ~7rr~ra~3t~n o~ ep ~ta~t l;7i~g t~!;e ~el4-1 cell lir~e ~elO-1 ~ell~ are tr~nsf3cte~ wieh the ~WA of the Icno~7n p~AV
2-3 A~ ep mutzr.t. pTAV2-3 has a ~Cra~es~irt~ muta.ior at positior lOg5 w~ereby all o~ the ~our ~ep proteius 2re i~activatr~d (c~. 7.~eilbronr~, ~, et al, ~. Virol. 64 tl~C~, 301~-3018~. Tlee Cll~ are ir~ected witn ad~o7rirus-2 ~fCI = 10-~0) . ~kerea~ter, the~ ~re induc~d 70ith lo~6 M
r7,=~.7rrl~th= ~.70re .
A~ter abou~ 30 h, part o~ tne c31~ E are ~ol~ecced and She entire cell DWA is iEolatec. It iB clea~ed b~ the restri~tior~ erlzywes X~al a~d DpnT, ~esp~cei~el~r, and analyzed in a ~;ouehern blo~. Por this purpo~e, 3~PP-labeled .
i lZ.f~PR.19~7 12:el1 HU13EF --'" --^ ---- ------''-^ "' 4 l4 NR.930 5.7/11 .. ~ , . I
Aa.V-DN~ is u~d as hybri~f7At7on sa~ale. rhe restrictiorl i anzy;r,e }D~al doe~ not cut ~AV-D~A, and the restriction en~yme Dphl does not cl~re a D~ re;~liaated in~o eukaryotes. ~arl irRf~ TAV2-3 ~ is obtained.
~u~ther~ore, the ~ o the non-collected cel ls is alte~na~ely ro~en and thawed ag ~1ell ~s ~ucjeoted to an u~trasounc t~eatment. ~ke R7lrFrng~Anf ~ ,tra~ed on ~elO-1 cells. Ille detectio~ o~ in~e4tioug A~VE i~ pur~ued ~y hybr7di~atiQ~ usin~ a 32P-laoeled probe speai~ic ~or rep-negative A~Vs. InCectious rep-regati~e ~AV paxticles are deteoted.
~he abcve example skow~ that xep-negative ~7~ m~ta~t~ Gan 'ae provided by t~e ceL1~ accor~i~g to the inv*ntior.
i~xz~ple ~: Frovi~icn o = a re3~ ga~ive AAV uuta3t u~
t~e Gall line EeC!Il~
He~ c~lls are co-trang~ectec witk the above pTAV 2-3 AAV-Re~ mutant and the exprasgi4~1 plasrnid EGO. rke cells are inected with adenovi7-us-2 (MOI = 1~-20). Therea~ter, tlLey are induced witk lO ~ ~10-7) M ~7~F~!=m~thA~one.
The cellE are tnÇ~ P~ up to ~he FUll ~ytoaathic e~tect -~ r~aus~d ~y adencviru~ ~ abo-at 4~ h~ and then l-nl ~ ~rtati by Prie~-~au ly~is and ultrasoun~ trea~ ant i~ a hypotonic bu~fer. ~he cell fl^~n~ -c are centri~uged of 1', the AAV
partiales ara C~ ~=; ned ir, th~ s~ a~n~nt . They al50 turn oue to be iriFctiou~.
~h~ above examrple e~ph28izes that rep-~egat7ve ~AV mutarltg can be provided hy cells accorcl~g to the invention.
!
, I
In The Matter of Canadian Patent ~rp1ir~1ti(1n-Applicant : Deutsches Kr~f~ ' ~ Stiftung Des o~r. Rechts; et al PCT Serial NO. : PCT/DE95/01429 Fihng Date : October 12, 1995 Title : I`~. ~ Of R.,~ dti.~ AAV Mutdnts And Cells Which Can Be Used Therefor Agent : Ivan St~ 'y Our File : 08-875722CA
Date : Apdl 14, 1997 The C, of Patents Ottawa-3Iull, Canada KlA 0C9 Madam:
NOTICE UNDER RULE 160(4) Until either a patent has been issued on the basis of the application or the apphcation is refused, or is otherwise abandoned and no longer subject to t, or is withdrawn the ~l is requested to lunit any: for furnishing a sample of the deposited material to an ' I l expert nominated by the C, in accoldance with Rule 165.
R~irertfil11y submitted, GOWLING, STRATHY & H~NDERSON, Agents for Apphcant.
Gowlin~, Strathy & Henderson lZ . f I rK . l9g ~ g HUBER ~A O 2 2 0 2 6 6 4 ï 9 9 7 0 4 1 4 NK . g3~ 5 . c /11 . 1 Pr~visics! o~ rep-~eSati~ ~AV ~utl-~t3 a3d cell3 w~ch can be used t~ere~
~he pregent invention relates to t~e prDVisioD. o~ . rep-negative AAV mutant~i and cell3 whJ ch can ke used there~or .
rAurthermor~, this invention ~oncerns an expressiQ~ plasmid u~ble for the prep~r~tiDn o~ the cells.
Ad6no-as3Dciated viru5es (AA~s) are r ing' e-strauded D~A
viruse~; hel nnri ~ to t~e ~amily o~ parvoviruses . AAVs ~e*d ~elpe~ TirUSes~ pa~ticularly ade~oviruses or herpesviruses/
~or the replicatlon therso~. In t~e absenoe of he~per viruses AAVs iLterrate into the host ~ell genome, particula~ly at a speci~ic s ' te D~ cn.romDson e lg and 17, respectively, Three viral run~tions wer~ rAl; ~ q on th* 4 . ~5~ ong, l~rLear geno~e or hurnan AAV-ty~e ~. ~he ~45 bp lcrg "ir.verted repeat=A~! serve as r~r1i~A~ n origi~ and as ois ~ignals ~or iut*gratiQn a~d packaging. Ihe c~;.a gene cDdes ~or three structural protein~ and the rep gene for a ~amily o~ lti~ nr~ihn~l regulator p~oteins. Iqle mRNAs for Rep 78 ~nd its C terrrl~al-sp~iced version Re~ ~B start at ~he p5 p:~onLoter Two ~ terminel t u3cated versions o~ Rep ~7~
and ~*~? 68, llantc'y Rep 5;~ an~ Rep 40, respec~ively. are expreAsed under the cortral o~ thA pl9 prornot*r. R*p DrOteins ~re n*c*ssary ~o~ th* ~NA r*Flication o~ AAV. m addition, they are ra~Iuirad ~ar the oerle reoulatiDn a~ AAV.
AAVs suppress the tu~or ~evelooment in animal5 Fur~he~no~e, they suporess ~he c*l~ tranS~OrmatiQn aaused ~y onGOge~es as well as the induced r)NA ampli~icati4r. In addltion, AAVs have a~ anti-proli~erati~-e e~ec-iveness.
Re~ proteins o~ AAV are held r.DArpf~ e f or the a~ove acti~iti*s. ~owe~rer, an ~qg~ Anr o~ these activities to th~ indi~itiual Rep proteins and domai~s ther*o~, respe~tively, do~s not exist. ~ut ~ch ~n a~sign~nen~ would~--i .
-lZ. ~PR . l9~ 01a HUBEFCA 0 2 2 0 2 6 6 4 1 9 9 7 - 0 4 1 4 NR . 9 30 5 . 3/11 - 2 - !
.
be r~ecessarv to ~e able to th~r~re~tic~ ly use ~A~7s. A
r~s; h~ y o achie~ ng this aasignment consists in the investi~ation o~ s h~vins mutations in the Rep protQins.
Many experi~:lents ~av~ been n~ade in thia respecc. ~o~ever, the provision o~ rep-r,egative ~ tarts wnich are f,rse~.
~rom wil d-tyoe .~AV, has not succesded S4 far. B-~lt tbe~ are o~ligatory ~or the above investigation~, There~ore, it is ~}~e object o~ the present invention to provide a mean6 by which rep-ne~ative AAtt mutants can be obtained withcut the ~ove d~ baake.
Ac~or~ing to ehe inv~ntior. this is ach~eved by ~he ~ubject tnatters defi~ed in the clai~
I'hus, ~he s~aj ect ~na~ter o_ the invention ~elates to cells which staoiy express the A~.V Rep Protei~s 7a and 52 s~ ~ell as 40 and/or 68. Cells ~hich ~xpress the ~ep proteins 78, 52 ar.~ ~ are pre~errec.
Cells accor~ing .o the invention car. he prepared as usual.
Cells o_ the ~c~own lire ~;eMl are avarably use~ as iriti~l mater~al ~c, ''Sth ~vc~rirl~ Workshcp", Ch~ystal Riv~, ~lorlda, U.5.A., ~ovember 10-14, 19g3)~ Thege cells ~p~ess th~ 7 R~p proteina ~8 ard ~2, ~he Re~p 78 e;~?rYsaion beirlg controlled by dexamethasone- i~ducibls ~Tr~-L~rR Cellg of ~e~?~ are tran~ected ~ith an expx~ssion plasmid ~odir.g _or Rep 4~ and/or one c4ding ~c~ Rep 68 and an expresaior~
plag~id whic:s ~odeg _or both ~p proteins, respectively.
exp~esaicn plasmid for Rsp ~0 is used pre~erably, the expressio~ pla~mid pCMRep 4C bei~ r~c;~lly preferre~. Tt contair~s ehe D~A coding ~or ~ep 4~ ~et~oeen restrictior.
sites ~rotl ar~ X~al c~ the ~nown ~ector pX~X-2-XI ~
Rit~ne~ . et al., ~5ethod~ ~ol. C~ Biol. 2 ~l991), 176-181). pCMRep 4~ was ~eposited wit~ the I~SM ~Ce~mar. Type t~oll~ctio~ o~ Mic r 4o ~ isn~s and ~ell C!uleurea Gmb~) tmder DSM 9491 on Oct. 7, l9g4 and under DS~ 9488 on Oct l9, 1Z.~PR.1g~7 12-0EI HUB~A 02202664 1997 04 14 NR gs0 5.4i~l 1 l9g~. It also oelorhgs to the s~iect matter o~ tk~
in~s~tioIl.
Tke cells Gbtained ~3y mea~s or LLCI~ LiQn o~ pCMRep ~4 stably expres6 the AAV ~ep pr4teins Rep 78, Re~ 5Z ancr Rep 40 These cells -~ers deposited as ~ell line ~e 10-1, Ee ~2-2 and E~e 5-5 with che DS~q u~der ~SM ACC2193, DSM AG~2192 and I~S~q ACC2131, respe~ti~rely, or. Sep. :~&, 1994 Furthermore, the cellY ~ere d~posited as :ell lire KeCMlg with the DSM under ~Sgl ACC21aS on A~g 3~, 19g4 The a~ove cells also ~e'ong to th6 a 6ubje~t matter oi~ the irven~ior Another s~7ect IDatter o~ ~he irvention relates to a pro~e6s ~C~ pro~riding re~?-nesative ~AV mutant6 Such a ~roces6 ~omprise6 the i~ollcwing proces~ing tep~:
(a~ trar.s~ection o~ ceil ~ rr~7:n~ to the inver.~ion ~7it~
the DNA c~ a rep-nègative AAv ~utant, ~! treatme~.c c t~e ~a~s~ec~ed cells o~ (a~ with a mea~7 eraoling an AAv replicatiou, p~rti~7ula_1y an A~l~-helper ~ irus, a~d (c) isolatio~ Qr ~he rep-regative AAV mucants obtai~e~
Ib) ln ~ pre~erred ' ~ , ca-tr~s~ectior with Ar expression 7~7asmid coding for a glucocorticoid reoeotor takes place il procesEir~ step ~a) . A p~rson ~killed in the a~t is ~ami' iar ;herewit~ ~or example, he kn~ws the e~YpresEicn plasmid HG0 (c~. Rumar, V, et al, Cell 51 (19E'7~, 941-9~1) Fnrther~ore, proCessing step ~a) il~lplieS tEle in~e~ion o~
oells a~:ordi~g to the irLventior. ~vith a ~ep-neg~ti~e AAV
mutant _n addition, the person ski7 led in the art iE
~arn~iar w~th all t~q~.nique~7 ne~es~7ary to carry ont the a~ove proces~ing steps. Re~ererce is made ~o MaDiati~ et ~1" hlol~.11r~r Cloning: A la}~oratory manlla' ~19~2), Co_d Sp~i~g H~rl~or, ~7eu YQrk, 3~y way 0~ Sl~rrlPmP~ ~
':, _ I
lZ.~lPR. 1397 12: E11 HU~EFCA 0 2 2 0 2 6 6 4 1 9 97 - 0 4 - 1 4 NR. 930 5. 5/1~
, . ' I
_4- !
The ex~aressior~ NA of a rep-negative A~V mutant" cosprises an ~ genome Pptionally present in a vector and having n~ in t~l6 rep ger~e. Such ~ nn~ r~ay be partiaularly ~eleeiona, ~ns~:~tion6 and~or substi~utions o~
one or more nucleotides. Tb.e AAv-r~A mqy als4 'oe a de~6tion o, the e~tire region ~oding for Rep. ~n ad~ition, the l~:~A
coding for Rep rita~r ~cartially or ~ully 4e replaced by a DNA
coding +or a ~oreigr protein ~ arei~ ~eptide) G~d ~y a DNA
coding for an ~n~; ~Pn~ ~A, ~es4ectively. The ~oreign ~rotein (+~oreign peptide~ aIld the I~Rn~; ~Pn~e~ll ~'~A, res~e~tively, ia prerera~'oly suita~le ~or gene-tke~apeutic asu~es. ~husl ~he ~co_essian ~rep-negative ~V mutant also i~Lplies tke ~err. "rep-negG~ive A~V vector~.
M.oreover, the expres6ion "DN}~ o~ a rep-ne~ative A~V T~tant"
~lso camprises a ~NA ~hich in ~ddition to the aoove-Tn~ =d ~s~tR~;~n~: includeg further mu~ation6 in otker AAV~ A regions . These may be e . g . t qt; ~ln~ iTl the cap oene. ~n ~uch ~ ca9e, ~ t is re~uired ~hat an P~rc~ hl P
P.AV cap ~ere ia prese~t in tAe cell3 ac40rdirg to ,,~e inveTlt~ on. It m;~y be in6erted by means enabling the AAV
rep~ioation, e.g. the AP.V helper TJiru~. The person skill6d i~ the art i~ familia~ with methods OT- inserting a ~Av cap gene, e . g . in an ~V helper virus .
~he expression "A~V heiper vir-~6~ compri6es viru,es which en~ble a replication o~ AAVS, Thes6 viruse6 l_e pa~ti4~1~rly ad6no~rirus~ sucn as adeno~ u~-z ard he~p~svi~uses .
13y means o+T the present in~rention it is possi'ole to prepare ~ep-s3sga~ve AA~ ItL~ta~ts. ~hey are fr~ f~om wi:Ld-c~p6 AAv, since ~ ' 'Tl~t~n OC~lT~rpn~c as h4rpf-T~ ir, t~e t-;~Tl~ e~pression of ~ep pr4teinE in c~lls, are avoided.
Thu~, the pres~IIt in~r~ntio~ ~ep~egent6 the basis ior restricting the acti~rit~ e~ ~s~ri~sPd to the ~ep proceins ta indi~idual Rep proteins and don~irs thereo+, resp6ctively.
I
lZ. I~PR. 19~77 lr: 01 HUBEI~A O 2 2 0 2 6 6 4 19 9 7 ~ 0 4 14 NR . 930 5. 6/11 -- .7 --Th~ s makes possible tb ~nvestigate in ~etail the mode o~
actio~ of A2.7r as tumor-sup~ressi7~-e r7~in~7r~ hich is ;nrliqr-,nq~hl-~ ior t~e use oi AAV in t~e t_eatment o$
tumo~;. Fu3:~he~o~e, ehe preserc invqr.tion o~ers.
~?ossihilitv of erovidin~ rs~n~h; ~r~ ~vs which qa~ be us=o as viral vectors ~o~ cene therapy. ~ep-~e~ative ~æv mtltants Ar~r~rr~;nn to the invention :r~ay bear ~enes ar~ gere s2ctions, respectively, usable ~or thiA p~rpose, ~ney ~ay be locatec~. ~articularly in the -ep gene an~cr cap ~;tene.
Thue, the presert inver~tion represent a break-through i~or the p7-r~r~rr~; nr of veceo~s usahle for ce~e cherapies.
_~ ~rie~ de 7cripeio~ c the dra~lrl~:
Fig. 1 shows a diagram o~ the Rep-cocillg DNæ in tke pC~ep40 expressior. p zs~.id. The start-.~l'G tri~le~t an~ the t~7~A~;nn codon ~,A are ircicated, ~hey co~re~pond ~o those oi t~e ~oild-t~pe-AaV ger.ome. The intron ~posieior.:
190~-2227~ is removed ~cy site-directed ~nutagenesis, so chat ~eo ~0 can be expressed witho~t splici~g.
The ir,vention is r~Ynl ~ i n~ s by the examples .
E~ample ~- ~7rr~ra~3t~n o~ ep ~ta~t l;7i~g t~!;e ~el4-1 cell lir~e ~elO-1 ~ell~ are tr~nsf3cte~ wieh the ~WA of the Icno~7n p~AV
2-3 A~ ep mutzr.t. pTAV2-3 has a ~Cra~es~irt~ muta.ior at positior lOg5 w~ereby all o~ the ~our ~ep proteius 2re i~activatr~d (c~. 7.~eilbronr~, ~, et al, ~. Virol. 64 tl~C~, 301~-3018~. Tlee Cll~ are ir~ected witn ad~o7rirus-2 ~fCI = 10-~0) . ~kerea~ter, the~ ~re induc~d 70ith lo~6 M
r7,=~.7rrl~th= ~.70re .
A~ter abou~ 30 h, part o~ tne c31~ E are ~ol~ecced and She entire cell DWA is iEolatec. It iB clea~ed b~ the restri~tior~ erlzywes X~al a~d DpnT, ~esp~cei~el~r, and analyzed in a ~;ouehern blo~. Por this purpo~e, 3~PP-labeled .
i lZ.f~PR.19~7 12:el1 HU13EF --'" --^ ---- ------''-^ "' 4 l4 NR.930 5.7/11 .. ~ , . I
Aa.V-DN~ is u~d as hybri~f7At7on sa~ale. rhe restrictiorl i anzy;r,e }D~al doe~ not cut ~AV-D~A, and the restriction en~yme Dphl does not cl~re a D~ re;~liaated in~o eukaryotes. ~arl irRf~ TAV2-3 ~ is obtained.
~u~ther~ore, the ~ o the non-collected cel ls is alte~na~ely ro~en and thawed ag ~1ell ~s ~ucjeoted to an u~trasounc t~eatment. ~ke R7lrFrng~Anf ~ ,tra~ed on ~elO-1 cells. Ille detectio~ o~ in~e4tioug A~VE i~ pur~ued ~y hybr7di~atiQ~ usin~ a 32P-laoeled probe speai~ic ~or rep-negative A~Vs. InCectious rep-regati~e ~AV paxticles are deteoted.
~he abcve example skow~ that xep-negative ~7~ m~ta~t~ Gan 'ae provided by t~e ceL1~ accor~i~g to the inv*ntior.
i~xz~ple ~: Frovi~icn o = a re3~ ga~ive AAV uuta3t u~
t~e Gall line EeC!Il~
He~ c~lls are co-trang~ectec witk the above pTAV 2-3 AAV-Re~ mutant and the exprasgi4~1 plasrnid EGO. rke cells are inected with adenovi7-us-2 (MOI = 1~-20). Therea~ter, tlLey are induced witk lO ~ ~10-7) M ~7~F~!=m~thA~one.
The cellE are tnÇ~ P~ up to ~he FUll ~ytoaathic e~tect -~ r~aus~d ~y adencviru~ ~ abo-at 4~ h~ and then l-nl ~ ~rtati by Prie~-~au ly~is and ultrasoun~ trea~ ant i~ a hypotonic bu~fer. ~he cell fl^~n~ -c are centri~uged of 1', the AAV
partiales ara C~ ~=; ned ir, th~ s~ a~n~nt . They al50 turn oue to be iriFctiou~.
~h~ above examrple e~ph28izes that rep-~egat7ve ~AV mutarltg can be provided hy cells accorcl~g to the invention.
!
, I
Claims (12)
1. Cells stably expressing the AAV-Rep proteins 78 and 52 as well as 40 and/or 68.
2. Cells according to claim 1, characterized in that they stably express the AAV-Rep proteins Rep 78, 52 and 40.
3. Cells according to claim 2, namely the cell lines He 10-1 (DSM ACC2193), He 22-2 (DSM ACC2192), He 5-5 (DSM
ACC2191) and HeCM1g (DSM ACC2185).
ACC2191) and HeCM1g (DSM ACC2185).
4. Expression plasmid, namely pCMRep 40 (DSM 9491; DSM
9488).
9488).
5. A process for the provision of rep-negative AAV
mutants, comprising the following processing steps:
(a) transfection of the cells according to any one of claims 1 to 3 with the DNA of a rep-negative AAV
mutant, (b) treatment of the transfected cells of (a) with a means enabling an AAV replication, particularly an AAV helped virus, and (c) isolation of the rep-negative AAV mutants obtained in (b),
mutants, comprising the following processing steps:
(a) transfection of the cells according to any one of claims 1 to 3 with the DNA of a rep-negative AAV
mutant, (b) treatment of the transfected cells of (a) with a means enabling an AAV replication, particularly an AAV helped virus, and (c) isolation of the rep-negative AAV mutants obtained in (b),
6. The process according to claim 5, characterized in that a co-transfection takes place with an expression plasmid coding for a glucocorticoid receptor in possessing step (a).
7. The process according to claim 5 or 6, characterized in that the DNA of the rep-negative AAV mutant of processing step (a) includes one or more deletions, insertions and/or substitutions in the rep gene.
8. The process according to claim 5 or 6, characterized in that the rep gene is deleted in the DNA of the rep-negative AAV mutant of processing step (a).
9. The process according to claim 5 or 6, characterized in that the rep gene is at least partially replaced by a foreign gene in the DNA of the rep-negative AAV
mutant of processing step (a).
mutant of processing step (a).
10. The process according to any one of claims 5 to 9, characterized in that the AAV helper virus is an adenovirus or herpesvirus.
11. The process according to any one of claims 5 to 10, characterized in that the DNA of the rep-negative AAV
mutant of processing step (a) includes a further mutation in the cap gene, with the proviso that the means enabling the AAV replication, particularly the AAV helper virus, contains an expressible cap gene.
mutant of processing step (a) includes a further mutation in the cap gene, with the proviso that the means enabling the AAV replication, particularly the AAV helper virus, contains an expressible cap gene.
12. rep-negative AAV mutant, obtained by the process according to any one of claims 5 to 11.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP4436664.7 | 1994-10-13 | ||
| DEP4436665.5 | 1994-10-13 | ||
| DE19944436665 DE4436665C2 (en) | 1994-10-13 | 1994-10-13 | Provision of rep-negative AAV mutants and cells that can be used for this |
| DE19944436664 DE4436664A1 (en) | 1994-10-13 | 1994-10-13 | Cells that stably express specific adeno-associated virus Rep proteins |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2202664A1 true CA2202664A1 (en) | 1996-04-25 |
Family
ID=25941026
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2202664 Abandoned CA2202664A1 (en) | 1994-10-13 | 1995-10-12 | Preparation of rep-negative aav mutants and cells which can be used therefor |
Country Status (4)
| Country | Link |
|---|---|
| EP (1) | EP0785991A1 (en) |
| JP (1) | JPH10507352A (en) |
| CA (1) | CA2202664A1 (en) |
| WO (1) | WO1996012010A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5866552A (en) * | 1996-09-06 | 1999-02-02 | The Trustees Of The University Of Pennsylvania | Method for expressing a gene in the absence of an immune response |
| CA2264482A1 (en) * | 1996-09-06 | 1998-03-12 | The Trustees Of The University Of Pennsylvania | An inducible method for production of recombinant adeno-associated viruses utilizing t7 polymerase |
| IT1297074B1 (en) * | 1997-11-21 | 1999-08-03 | Angeletti P Ist Richerche Bio | HORMONE-DEPENDENT FORMS OF THE REP PROTEIN OF THE ADENUS ASSOCIATED VIRUS (AAV-2), DNA SEQUENCES CODING FOR THEM, VECTORS THAT |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU8200191A (en) * | 1990-07-09 | 1992-02-04 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | High efficiency packaging of mutant adeno-associated virus using amber suppressions |
| ES2220923T3 (en) * | 1993-11-09 | 2004-12-16 | Medical College Of Ohio | STABLE CELLULAR LINES ABLE TO EXPRESS THE REPLICATION GENE OF ADENO-ASSOCIATED VIRUSES. |
| EP0733103B1 (en) * | 1993-11-09 | 2004-03-03 | Targeted Genetics Corporation | Generation of high titers of recombinant aav vectors |
| US5693531A (en) * | 1993-11-24 | 1997-12-02 | The United States Of America As Represented By The Department Of Health And Human Services | Vector systems for the generation of adeno-associated virus particles |
-
1995
- 1995-10-12 CA CA 2202664 patent/CA2202664A1/en not_active Abandoned
- 1995-10-12 JP JP8512845A patent/JPH10507352A/en active Pending
- 1995-10-12 EP EP95934604A patent/EP0785991A1/en not_active Withdrawn
- 1995-10-12 WO PCT/DE1995/001429 patent/WO1996012010A1/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| WO1996012010A1 (en) | 1996-04-25 |
| EP0785991A1 (en) | 1997-07-30 |
| JPH10507352A (en) | 1998-07-21 |
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