CA2216871A1 - High efficiency ex vivo transduction of cells by high titer recombinant retroviral preparations - Google Patents

Abstract

Compositions and methods for the efficient ex vivo introduction of nucleic acid into T cells, non-dividing cells, and cells resistant to standard transduction techniques mediated by high titer recombinant retroviral preparations is described. The recombinant vector constructs carried by the recombinant retrovirus particles code for the production of one or more desired gene products from one or more corresponding genes of interest, at least one of the gene products having a therapeutic application. Upon re-introduction into a patient, the transduced cells produce a desired gene product in an amount sufficient to treat a particular disease state.

Description

W 096/33282 PCTrUS96/05638 HIGH EFFICIENCY EX ~VO TRANSDUCTION OF
CELLS BY HIGH 1'11'l~'R RECOMBlNANT
RETROVIRAL PREPARATIONS

Technical Field The presc~t invention relates genc, dl~ to, eco. . .l .i ~ retroviruses and genetherapy, and more specifically, to high titer l~cQ...kil~ retroviral particle plc~a~lions suitable for a variety of gene therapy apI!lir,~ti Back~lvund of the Invention Since the discovery of DNA in the 1940s and con~ g through the most recent era of recol~lbi~ l DNA teçhnology, s.~l,sl i. nl ;~1 r eseau ~h has been undertaken in order to realize the possibility that the course of disease may be a~e~;led through interaction with the nucleic acids of living olp,~n;~ ~ ~C Most I cce.~lly, a wide variety of mPthntls have been described for ~lt~o.ring or ~c~;~-P genes of som~tic tissue (a process so~ Ps Icrellcd to as "som~tiC gene therapy~), inr~ irtg for .~ r lc~ viral vectors derived from rcLIuviluses, adenovi-uscs, vaccinia viruses, herpes viruses, and adeno-~oc:-le~ viruses (see JoDly, Cancer Gene Therapy 1(1):51-64, 1994), as well as direct ll~u~rer terhni~les such as lipofection (Felgner et al., Proc. Nafl. Acad. Sci. USA 84:7413-7417, 1989), direct DNA
injection (Acsadi et al., Nafure 352:815-818, 1991), microprojectile bc,ll.l~dll,~
(Williams et al., PNAS 88:2726-2730, 1991), liposomes of several types (see, e.g, Wang et al., PNAS84:7851-7855, 1987) and ~ ion of nucleic acids alone (WO 90/11092).
Of these techniques, ~ c;Co~ ell ~vi, ~I gene delivery methf rl.~ have been mostextensively ~tili~P,(l, in part due to: (1) the Pffiri~nt entry of genetic material (the vector genome) into replic~tin~ cells; (2) an active, Pffir;~nt process of entry into the target cell nl-cle~l~; (3) relatively high levels of gene cApl~ion; (4) the potential to target particular cellular subtypes through control of the vector-target cell binding and the tissue-specific control of gene eA~Ic~ion; (5) a general lack of pre-PYi~tin~ host ;.. ~.;ly, and 30 (6) substantial knowledge and clinical experience which has been gained with such vectors.
Briefly, retroviruses are diploid positive-strand RNA viruses that replic~te through an integrated DNA interrnP~i~te In particular, upon infection by the RNA virus, the retroviral genome is reverse-transcribed into DNA by a virally Pncoded reverse transcriptase that is carried as a protein in each retrovirus. The viral DNA is then 35 integrated pseudo-randomly into the host cell genome ofthe infecting cell, forming a "provirus" which is inherited by d~lghter cells.
Wild-type retroviral genomes (and their proviral copies) contain three genes (the gag, pol and env genes), which are preceded by a p~r~ginp signal (~), and two long W 096/33282 PCTrUS96/05638 t~rmin~l repeat (LTR) sequenres which flank both ends Brie~y, the gag gene ~ncQ~es the internal structural (mlcleor~rs;~l) p-ùlei~ls Thepol gene codes for the RNA-dependent DNA polyl-le.~se which reverse ~ ;bes the RNA ~;enrme, and the env gene ~nr~o(l~s the IcL-uvi-~l envelope ~Iy~;~protcills. The 5' and 3' LTRs contain cis-acting el~m 5 n~Gf~.~;S~.y to promote l,; ~ n and polyadenylation of reLIuvi~l RNA
qc~ont to the 5' LTR are seq-l~nres n~ce~ for reverse L-~ s~ ,Lion of the ~en~rr~ (the tRNA primer binding site) snd for effi~nt f -~ ~p;~ of l ~ll u ~ l RNA
into particles (the y seq~l~once) Removal of the pa~ ging signa1 p- cvc..Ls ~nr~ps;tl~tirn ~ng of r cL.ovi-~l RNA into inf~ctioll$ virions) of ~nnm;r, RNA, ~hhrJ~lgh the 10 resllltin~ mutant can still direct ~Lhc:~;s of all p-uLcil~ enrode~ in the viral 5~n~m~
P~PGQ h;~ l ~cL-uvi uses and various uses thereofhave been ~lesrrihe~l in ~u~e ~uS
references in~ for ~Y~mplP; Mann, ef al. (Cell 33:153, 1983), Cane and Mlllli~n (Proc. Nat'l. Acad. Sci. USA 81:6349, 1984), Miller, etal., Human Gene Therapy 1:5-14, 1990, U S Patent Nos 4,405,712; 4,861,719; 4,980,289 and PCT Applir-~tif~n Nos WO
89/02,468; WO 89/05,349 and WO 90/02,806) Briefiy, a foreign gene of interest may be - incorporated into the r cL o~ s in place of a portion of the normal retroviral RNA When the rcLlovil~ls injects its RNA into a cell, the foreign gene is also introduced into the cell, and may then be il~Lcgl~Led into the host's cellular DNA as if it were the I cLlovil us itself Expression of this foreign gene within the host results in c,,~ ,.ûn of the foreign protein 20 by the host cell One disadvantage, however, of rcco.. l .h.~ .~ retroviruses is that they prinrir~lly infect only replic~tinp cells, thereby making Pfflrient direct gene ~ re~ rliffir,lllt or impossible for cells ~,L~cl~ ed as largely non-replir~tin~ ition several other types of cells inr~ in~ T cells, B cells, monocytic cells and dPn~1ritir cells have tr~riitirJn~lly been rliffirlllt to tr~ncduce by retroviral vectors, even when stim~ ted to replic~te- This was particularly true for plimaly cells Indeed, some sriontiete have su~este~l that other, more ~ffi~i~nt methnds of gene ~ rel~ such as direct ~ minietration of pure plasmid DNA, be utilized (Davis et al., Human Gene Therapy 4:733-740, 1993) to introduce nucleic acid moleclllP,s into such cells In order to increase the efficacy of recollllfinallL .e~.uvil .Ises, the mPthods which have been sll~eted have prinrir~lly been aimed at in~ucin~ the desired target cells to replicate or to replicate more Pffir;~ntly, thereby allowing the retroviruses to infect the cells Such methods have inrlllde-l, for PY~mple rhemic~l tre~tmPnt with 10% carbon tetrachloride in mineral oil (K~1P1~O, ef al., Human Gene Therapy 2:27-32, 1991)However, such terhnitlues are not pl~rt:lled for use in ex vivo techniques dPeignP~d to introduce nucleic acid moleclllP,s encoding therapeutic gene products into animal cells For T cells, various methods of in vitro non-specific stim~ tion (such as IL 2, pokeweed mitogen, and anti-CD3 antibodies) can be quite Pffiri~nt in intluring replir,~tion However, W 096/33282 PCTrUS96/05638 .
.liffic~.ltiPs rPtn~inP~d in obtaining Pffir;~rlt tr~ncd~lctiQn with mPths-lc con~aLil~le with clinical and cc",---le,.,;al use.
Fffiri~nt gene ~ rèr into anim~l T cells and non-rP~lir~tin~ cells h-as proven .1iffirll1t due to a variety of factors. Cu~lelllly used methnrl$ of IcLluvil~l tr~nc~urtinn into S such cells have a llulllbei of practical 1imit~tinnc Such limit~tionc are collll ~ullded by t-h-e rela~ively low titers generally obLa,l~ed with most retroviral vectors, typically in the range of 10~ to 106 infectiQllc virions per millilitPr.
The range of host cells that may be i..re~i~ed by a retrovirus or lelluv~l vector is delellllined in part by the viral envelope protein. Thelerole~ a lack or defiri~pncy ofthe 1 cel)LOr for the given envelope protP~n would limit tr~ncdllctinn Pffiri~nry In nd~lition~ a lack ofthe re4~c;le cellular factors involved in viral binding, penellalion~ ro~ ofthe retroviral vectors, viral replir~tion or il~leglaLion would limit tr~ncd~lcti~n PffiriPnry. In adl1ition the low titers of available vectors have npcec~ l ed methorlc using co-cultivation with vector prod~1rin~ cells. Alternatively, it has been nPc~Pc~ y to add large volumes of vector plé~ lions to the culture met~ m co~ g the cells to be tr~ncd~ced to achieve useful tr~ncducti~n freq~Pnries This leads to a disLu-lJallce ofthe culture cQntlitiQnc for the target cells. These and other problems are addressed by the instant invention.
Tt is the objeGt of ~,he preser.t ~ t~ p~r8~ide ~ .t~!XV~ ~ nl~;h~ 5 for using compocitinnc of high titer ~eco~hi~ l rèLIo~ particles to deliver vector con.,LIu~
enr,o~in~ genes of interest to T cells, non-replir~ting cells, and cells lesi~l~ll to sL~ld~d tr~ncd~cfir~n terhni1~lPc The tr~ncd~lced cells may then be re-~d~ p~ ed to the patient by standard techniques, e.g, il~lla~ellu.ls infil~;nn to achieve a desired ILelape~llic benefit.
Su,.... ...",~,y ofthe Invention The present invention provides compositir~nc and mPthnrlc for tr~ncd~lring T cells, non-dividing cells, or cells resistant to sl~ld~d tr~nc<luction terhn;ques colll~ lng oblaillil~g a population of T cells, non-dividing cells, or cells 1~ ~ll to standard tr~ncducti~n terhnirlu~Ps with a plep~ alion of high titer leco~ ~ ~hj~A~II retroviral particles subst~nti~lly free from co.~ l;on with repiir,~tion cc,ll.pelelll retrovirus, wherein the reco-.ll~ill~ll lèlluvil al particles carry a vector construct encoding a gene of interest.
In another aspect of the invention, an in vivo delivery vehicle cnmpricing tr~ncpl~nt~h'~ T cells, non-dividing cells, or cells resistant to standard tr~ne~luction techniques which express a thel~eulically effective amount of a gene product encoded by a gene wLeleill the gene does not occur in T cells, non-dividing cells, or cells resisl~ll to standard tr~ned~lr,ti~ n techniques or where the gene occurs in T cells, non-dividing cells, or cells rèsi~kull to standard trAnecluction techniques but is not c,~lèssed in them at levels which are biologically ,ci nificAnt or wherein the gene occurs in T cells, non-dividing cells, or cells resistant to standard tr~ncdllction techniques and has been modified to express in T

W 096/33282 PCTrUS96/05638 cells, non-dividing cells, or cells re~ t to standard tr~neductinn terhniquf~e and wherein the gene can be mnflifif~d to be e,~lf ssed in T cells, non-dividing cells, or cells le~ t to standard tr~ncfluction te~ h.~ es is provided.
Within another f~mho-1imf~nt ofthe invention whel~l the vector construct enr,of~1es a 5 molecule se,1ected from the group co~ P of a protein, an active portion of a protein and a RNA mnlec11le with intrineic biological activity. The protein or active portion of a protein issP1ectedfromthegroupcf~lic;~ ofa~,~ul~.~, a cc~ ysl;~
factor, and a hnrmf nf~
Within still another emboflimf~nt the pop~ tif~n of T cells, non-dividing cells, or 10 cells l~,S.~ll to :,~ndald tr~ncduction terhniquf~s is obt~ined from an animal. In another emborlimf~nt the animal is a human ~. .fr~ from a disease chara.;~ d as a disease sf~1ected from the group conc:el;..g of a genetic disease, cancer, an infectimlc disea_e, a f~1egf~-nPrative disease, an i.. nn.. ~1O~y disease, a cardiovascular disease, and an ~loi..~ e disease.
Within still another embodiment mrthf fle are provided for t,~g ~ f~ere such as a genetic flicf~ef-, cancer, an infrctio11c disease, a degen~ ive disease, an i~ oly disease, a cardiuv~s~ul~ disease, or an ~uto;~ e disease by ~fl~ ing to a patient a cnmrositinn or re-introfl11r,tif n of a ~ .nl-e~;r-~lly effective amount ofthe poplll~tinn of tr~ncfduced T cells, non-dividing cells, or cells ~ to s~ildald tr~nefluf tinn t~ ' ques 20 In another another emboflimf~nt the T cells, non-dividing cells, or cells lt;~ to standard tr~neduction terhniq11f s are eyr~nfled in vif~o prior to re-introduf tion of the cells into the patient.
In other aspects of the invention the tr~neduced T cells, non-dividing cells, or cells lesisl~ll to standard tr~neduction terhniques and compositions oftr~ncduced T cells, non-25 dividing cells, or cells r_~ t to standard tr~nc~lucfinn terhniqupe Pnf oflin~ a gene ofinterest are provided. The envelope protein of the high titer I eco. ~ .1.; . . - ~ .1 1 ell UVil ~I p articles is an envelûpe protein s~1ectf d from the group concietinF~ of a l~lUVi~ amphotropic envelope protein, a retroviral ef U~IUp c envelope protein, a It;lluvil~l polytropic envelope protein, a retroviral xenotropic envelope protein, a gibbon ape lellkpmi~ virus envelope 30 protein and a VSV-g protein. Other retroviral envelope plUt~ls known to those of skill in the art can also be used.

Definition of Terms The following terms are used throughout the sperific~ti-on Unless othervvise 3s infiif;~tefl, these terms are defined as follows:
"Event-specific promoter" refers to ~ s-;li~ ~ional promoter/Pnh~nr,er or locus dPfining el~pmpnte or other f-1~mf~ntc which control gene ~,A~ression as fliecussed above, whose ~ scli~lional activity is altered upon response to cellular stimuli. Repl~sf lll~live W096133282 PCTrUS96/05638 .
PY~mr1~s of such event-specific promoters incrude thymidine~kinase or thy~nidylate ~yl~Lase p,olnolel~, a or b i~lelreloil plon~ole.:i and promoters that respond to the prcscnce of hn~ ---nl-~s (either natural, synthetic or from other non-host O~,,AI~ C.,~C).
nTissuc-s~eci~c p.~nlole~" refers to l,~.scliplional p~ul~loLer/PI~h~l~c~ or locus S dPfinin~ Pl~mPntQ- (eg. Iocus control plemPntc)~ or other el~ which control gene e~ c~s;on as ~ above, which are pler~,.Lally active in a limited ~un~l~e~ of L~.llalOpoiclic tissue types. R~ /h5ofsuc~ llalu~oietic tissue-s~e_ifi~
pl olllolel ~ include but not limited to the IgG p. .~I.loler~ a or ~ globin promoters, and T-cell It~e~)lor p~ le .
"Tr~nQ~ ction~l involves the ~Q-Q-oc ~I;on of a rerli~;~tiQn dcre.;livt;, ~ecc ~ h retroviral particle with a cellular rece~lor, followed by introtl~lction of the nucleic acids carried by the particle into the cell. "Tl~nQ-r~c~;onH refers to a method of physical gene ~r~re wl-er~ no lCIlOVil7~l particle is employed.
"Vector constructn, "lc~ ] vector", "rccci...h;.~ ~I vector", and "reco..-l; ~A~15 retroviral vector" refer to a nudeic acid construct capable of dhc~ling the ~ les~ion of a gene of interest. The retroviral vector must include at least one ll~lscliplional pl~,l"olel/P-nh~n~pr or locus ~lPfining Pl~ompnt(s)~ or other elP!mentQ which control gene c,~lcs~;on by other means such as alternate spli~ing nuclear RNA export, post-Il~ .Ql~;rln~ ifirAtion of...~LPIlp~r, or post-t~uL5~ ;0nal mn-lifi~tinn of protein~ In 20 ~d~lition the retroviral vector must include a nucleic acid mo~cc~lle which, when s~-;bed, is operably linked to a gene of interest and acts as a ~ ;on ;~ iOI~
seq~l~nr,e Such vector constructs must also include a p~r.l~n~ signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding sites al~plc p-;ale to the retrovirus used (ifthese are not already present in the retroviral vector) 25 Optionally, the vector construct may also include a signal which directs polyadenylation, as well as one or more restriction sites and a ll ~ ;on te~ Al ;nn seq~lpnre By waylplc~ such vectors will typically include a 5' LTR, a tRNA binding site, a p~rl~Agin~
signal, an origin of second strand DNA ~..II.e:,;s, and a 3' LTR or a portion thereo~ ~
order to express a desired gene product from such a vector, a gene of interest Pnr,o~ing the desired gene product is also inrl~ldecl A "RNA molecllle with intrineic biological activity" in~llldes ~nfiepnee RNA
molecules and ribozymes and RNA molecules that bind proteins As used herein, "cells resistant to standard trAneduction terhniq~les~ are cells which, in the presence of ~t;co---~ retroviral particles according to the invention which have titers of about 106 cfu/ml, as measured on a standard titering cell line such as Hl 1080, trAned~ce fewer than about 5% of the cells Such cells may include normal cells as well as those which are ~lieeAee~l such as tumor cells and; r~led cells, among others W 096/33282 PCTrUS96105638 A '~p~ ion~ of high titer ~cco...l~ retroviral particles refers to a liquid or Iyophili7rd composition co...~lisiilg such particles Preferably, the plt:p~ ion is equivalent to a phArmAce~ltirA1 comros;tinn in terms of its co~ ent~ but, as those in the art will appl~.,ial~;, when ~r~ lion is to cells other than for later human ;vl ..;.~ lion, such 5 prep~Lions need not be of phArmAceutir~l quality, and may in fact c~ e only crude, high titer relluv~l vector sllp~ produced in acco,-lance with the mr-tho~s desrrihe~
here~
The term "T cells, non-dividing cells, or cells resistant to standard trAn~lucti~n terhn~ es" inr~ les T cells, B cells, monocytic cells, d~nt1ritic cells, nerve stem cells, liver 10 stem cells, i..~ l stem cells, bone stem cells, kidney stem cells, skin stem cells, hair stem cells, non-dividing stem cells, non-dividing pancreas cells, non-dividing kidney cells, germ cells and other cells re~;sl~ll to standard trAnedllctisn terhnirlllec Progeny cells and pr~u.~or cells to the above cell types are also e ~ro~. pA~ed by this term, inr~ ing T cell ple~;U~Ol:i and B cell p~e~;ul~ul~ As used herein, the terms "T cell precursor" and "B cell 15 precursor" refer to all pre.;ul jor cells that are co ;1l~d to the T-cell di~e.~ l i9n paLllw~y or the B cell di~er~ l ;r~n paLl.w~y, res~,e.;livt;ly, but exclude non-commited cells such as he --aLopoietic stem cells Numerous aspects and advantages of the invention will be a~p~ellL to those skilled in the art upon con~ ration of the following d~tAiled description which provides ill.. ;.~:.l;r~n ofthe practice ofthe invention.

Detailed Desc.;~,Lion ofthe Invention The present invention is based on the u..~,A~e-;Led discove y that recon~il-~-L
25 retroviral particles of high titer which carry a vector construct comrri~ing one or mûre genes of interest can be used ex vivo to Pffic;~ntly tr~n~dllr~e T cells, non-dividing cells, and cells which are resistant to standard l~n~d~ ;r~n terhn;lues As a result, lc;co~reLluvil~l particles accoldillg to the invention can be used for purposes of gene therapy and to tr~neduce cells rollllell~ con~ ered to be difficult or impossible to tr~neduce with a 30 lelluvilus A more thorough description of such reco...hin~ reLlovil~l particles, their production and par~ ing and uses therefore is provided below Generation of Reco.llbinallL Retroviral Vectors As noted above, the present invention provides compositions and methods comprising recf.l- hi~ ellovil~l partides for use in ex vivo som~tic gene therapy The construction of recûll-~i,l~l~ retroviral vectors and particles is described in greater detail in an applic~tion entitled "Reco...bina.~L Retroviruses" (U S S N 07/586,603, filed September W 096/33282 PCTrUS96/05638 21, 1990, which is hereby incol~ol~ted by lcrclci~ce in its entirety). Protll~ctiQn of tr~n~d~lction co~.pe~ cco.~ l retroviral particles is descAbed in U.S.S.N.
07/800,921 and U.S.S.N. 07/800,921, which are hereby il~ ol~led by lcrcrel ce in their entirety. In general, the lccc)l~hil-~.l vector constructs described herein are prel)a cd by 5 selecting a pl~cm;~ with a strong plo.~ol~, and applupliale r~str~ction sites for ~-sclliol of DNAseq~Pnr,P~ of interest dowl.sllc~ll from the prol..oler.
According to the invention, the lccoml,..~ vcctor construct is carnc~ by a rcco~ .l lelluvilus. Rcll~)vi,uses are RNA viruses with a single positive strand g~PnomP
which in general, are nonlytic. Upon infectinn~ the retrovirus reverse t~ sclibes its RNA
10 into DNA, formirlg a provirus which is inserted into the host cell g~ e. The lc~l~.vil~l gPnOmP can be divided collccplually into two parts. The Htrans-actingH portion CQI~ of the region coding for viral structural proleh~s, inr~ i~ the group specific antigen (gag) gene for synthesis of the core coat prolcills~ the pol gene for the synthesis of the reverse L,al~scli~l~sc and illLe~se el~ylllc~, and the envelope (env) gene for the synthesis of 15 envelope glycoploleil s. The "cis-nctirlg" portion col.~ of regions of the gennmP that is finally p~r1~g~d into the viral particle. These regions include the p~rt-~gin~ signal, long termin~l repeats ~LTR) with promoters and polyadenylation sites, and two start sites for DNAreplir~tinn The internal or "trans-acting" part of the cloned provirus is replaced by the gene of interest to create a Hvector constructH. When the vector construct is placed into a ce~ where viral p~c~gin~ proteins are present (see U.S.S.N.07/800,921), the h~ e~
RNAwill be p~rl~aged as a viral particle which, in turn, will bud off from the cell. These particles are used to tr~n-~dl3ce tissue cells, allowing the vector construct to il~le~ale into the cell ~enomP ~lthnugh the vector construct express its gene product, the virus callying it is replir~ti~n defective because the trans-acting portion of the viral genome is absent.
25 Various assays may be utilized in order to detect the presence of any replic~tion comretpnt infectious retrovirus. One plcrellcd assay is the eytrn~led S+L- assay described below.
In the broadest terms, the retroviral vectors of the invention conlplise a sc.i~lional prc,llloLel/Pnh~nr,~r or locus ~lPfinin~ Pl~m~nt(s), or other f~l.o.m~nt~ which control gene ~ lc:l~ion by other means such as alternate spliri~ nudear RNA export, 30 post-l,;.n~ ;Qn:~l mor~ifir.~tintl of ...ç5S~np~r, or post-transcriptional ms)tlifir~tion of protein. In ad~litinn~ the rcllu~ vector must include a nucleic acid molecule which, when tr~n~rribecl in the gene of interest, is operably linked thereto and acts as a translation initiation sequçnr,e Such vector constructs must also include a p~ ing signal, long terminal repeats (LTRs) or portion thereof, and positive and negative strand primer binding 35 sites applopli~le to the retrovirus used (ifthese are not already present in the rclluvilal vector). Optionally, the vector construct may also include a signal which directs polyadenylation, as well as one or more restriction sites and a tl~ n~l~3l ;on t~ . . .;. .i11 ;on sequen~e By way ~Y~mrl~ such vectors will typically include a 5' LTR, a tRNA binding W096/33282 -8- PCTrUS96/05638 site, a p~ gin~ signal, an origin of second strand DNA :iyl~Lhe;,;s, and a 3' LTR or a portion thereo~ Such vectors do not contain one or more of a c~mrlete gag, pol, or e7~
gene, thereby r~n~ rin~ them rep~ tirm illcolll})e~ell~. In ~d~lition~ nudeic acid m~ c~ s coding for a sPlect~ble marker are neither rt;~luiled nor yuerel~ed.
S l?lèrel~ed retroviral vectors contain a portion of the gag coding seq l~n~
prère~bly that portion which co...~.. ;c~s a splice donor and splice acceplor site, the splice ~cce~o~ site being positiQn~d such that it is located ~ , a~ 1,~;,ll~., fi~m~c gene of interest. In a particularly plerel~ed emhotlim~nt~ thegag L"~ls~; ip~ional promoter is position~d such that an RNA ~ scliyl inhi~ted thèueLunl co..~ the 5' gag llTR and the gene of interest. As an ~1l e~ 1 ;ve to the gag promoter to control e,~yles~ion of the gene of interest, other suitable promoters, some of which are described below, may be employed. In a~ itiOIl, slltf~.rn~te ~nhsln~rs may be employed in order to increase the level of ~ réssion of the gene of interest.
In ~ere~ed embo-limP!nte~ ofthe invention, rellovh~l vectors are employed, particularly those based on Mol~nP,y murine l~llk~mi~ virus ~oMLV). MoMLV is a murine .è~.uvi-~s which has poor infectivity outside of mouse cells. The related~mphotropic N2 rtl.~vi Lls will infect cells from human, mouse and other o~ nie~e Other p~ere~ed rehovi uses which may be used is the practice ofthe present invention include Gibbon Ape T ellkrmi~ Virus (GALV) ~Todaro, ef al., Virology, 67:335, 1975; ~llson, ef al., J'. Vir., 63:2374, 1989), Feline ~mmlmodefir;~nry Virus (E~IV) (Talbatt, ef al., Proc.
Naf 'l. Acad Sci. USA, 86:5743, 1984), and Feline T ~llkrmi~ Virus ~FeLV) ~Lepl cvelle~ ef al., ~ Vir., 50:884, 1984; Elder, ef al., J. Vir., 46:871, 1983; Steward, ef al., ~ Vir., 58:825, 1986; Riedel, ef al., J. Vir., 60:242, 1986), ~lthough retroviral vectors according to the invention derived from other type C or type D retroviruses or lentiviruses or spuma viruses (see Weiss, RNA Tumor Yiruses, vols. I and II, Cold Spring Harbor Laboratory Press, N.Y.) can also be generated.
A v~iel Iy of prolllotel:i can be used in the vector constructs of the invention, inr~ 1inF~ but not r~Pceee~rily limited to the ~iylu~ nvirus major ;...~ e earlyprulllc,ler (CMV MIE), the early and late SV40 promoters, the adenovirus major late 30 promoter, thymidine kinase or thymidylate synthase plOIl~OLel:i, a or b il.te,relun promoters, event or tissue specific promoters, e~c. PlOIllOtel~ may be chosen so as to potently drive high levels of ~ essiOn or to produce relatively weak c A~iession, as desired. As those in the art will appreciate, numerous RNA polymerase II and RNA polymerase m depçn~nt promoters can be utilized in pr~ctirin the invention.
In one embo~limrnt l eco.. .h;~ retroviral vectors com~ g a gene of interest are under the ~ scliplional control of an event-specific promoter, such that upon activation of the event-specific promoter the gene is expressed. Numerous event-specific promoters may be utilized within the context of the present invention, in~ r1in~ for eY~mplto, promoters W 096/33282 PCTfUS96/05638 _ 9 _ which are activated by cellular prol~feration (or are otherwise cell-cycle dep~n~lPnt) such as the thymidine kinase or thymidylate synthase promoters (Merrill, Proc. NatL Acad. Sci.
USA, 86:4987, 1989; Deng, etal.,MoL Cen Biol., 9:4079, 1989); orthe ~I;...~r~..;.
receplor pr~ll,oler, which will be l,;.,.cl~. ;pl;nn~lly active primarily in rapidly prolire~
5 cells (such as helll~lopoietic cells) which contain factors capable of activating h; ne~ l;nn from these pro.ll.alel~ prererelllially to express gene products from gene of illLeleSl, pl~l--UICil~ suc~ as the a or b ~Le~re O~J1U11~ which are ~Li~ lel when a cel~ is infPcted by a vi~us (Fan and lU~ni~tic EI~BO J., 8:101, 1989; fioodbol~rn, etal., Cell, 45:601, 1986); and promoters which are a~;Liv~led by the presence of h~s....ol-es, e.g, estrogen le~onse promoters. See Toohey et al., Mol. Cell. Biol., 6:4526, 1986.
In another embodiment, lèco...l~ A..l retroviral vectors are provided which coll-~-ise a gene of interest under the l.~uls~ lional control of a tissue-specific plo---oLer, such that upon activation of the tissue-specific promoter the gene is expressed. A wide variety oftissue-specific promoters may be utilized within the context ofthe present 15 invention. Repleselaliv-e ~y~mple~ of such prolllù~el:j inrlvde B cell specific promoters such as the IgG plclllotel, T-cell specific plulllOtel:j such as the T-cell rece~Lor plumoler (Anderson, et al., Proc. Natl. Acad. Sci. USA, 85:3551, 1988; Winoto and R~ltimore~
EMBO J., 8:29, 1989); bone-specific pl~lllulel~ such as the osteocalcin pl~Jllloler ~ ~rkose~ ef al., Proc. Natl. Acad. Sci. USA, 87: 1701, 1990; McDonnell, et al., Mol. Cell.
Biol, 9:3517, 1989; Kerner, etal., Proc. Natl. Acad. Sci. USA, 86:4455, 1989), the ~2 promoter, IL-2 receplor promoter, and the MHC Class II prc,lllùler, and hèmalopoietic tissue specific promoters, for ;~ e erythoid specific-transcription plul--o~e ~ which are active in ely~lluid cells, such as the porphobilinogen de~ ce promoter (Mignotte, etal., Proc. Nafl. Acad. Sci. USA, 86:6458, 1990), a or b globin specific promoters (van .~e.e.o.n~ ef al., Cell, 56:969, 1989, Folle:~er~ ef al., Proc. NatL Acad Sci. USA, 86:5439, 1989), endothelial cell specific plulllolel:i such as the vWfplc,l..ùler, m~g~k~ryoCy-te specific p-o---ù~e :i such as b-thrombo~loblllin and many other tissue-specific promoters.
Retroviral vectors according to the invention may also contain a non-LTR Pnh~nr~r 30 or promoter, e.g, a CMV or SV40 Pnh~nrPr operably ~Csori~t~pd with other PlPmPntc employed to regulate cA~lession ofthe gene of interest. ~lition~lly, retroviral vectors from which the 3' LTR Pnh~ncçr has been dPlete~l, thereby inactivating the 5' LTR upon integration into a host cell ~PnomP~ are also contemrl~ted by the invention. A variety of other PlPmPntC which control gene c A~lession may also be utilized within the context ofthe 35 present invention, inrl~ ing for example, locus-dP~ninF~ ekpmpntc inr.lnrlin~ locus control regions, such as those from the b-globin gene and CD2, a T cell marker. In addition, PlemPntc which control CA~leS:jiOn at the level of spli~ nuclear export, and/or l,nn~ n may also be inrl~lded in the retroviral vectors. Representative eY~mples include the b-W O 96/33282 PCTrUS96/05638 globin intron sequence~e, the rev and rre plPmpnte from HlV-l, the conetitlltive ~ ~oll elemPnt (CTE) from Mason-Pfizer monkey virus (MPMV), a 219 nllcleotirlp~ seq~lenre that allows rev-independent replir~tion of rev-negative ~V proviral clones, and a Kozak eeq~Pnce Rev protein filnctigne to allow nuclear export of Imerliced and singly spliced 5 H~V RNA molccl~lPs The MPMV elemPnt allows nuclear export of intron- c~
mRNA. The CTE Pl~mPnt maps to MPMV mlrleotid~ps 8022-8240 a ~Bray, ef al., Bioch- ".,~lr~, 91:1256, 1994).
In another p~crc~cd emhor1impnt the retroviral vector cc ..~ e a splice donor (SD) site and a splice ~cceptor (SA) site, wLelcill the SA is located up:iL Ca-~l ofthe site where 10 the gene of interest is i.~sc,Led into the reco...l~ ..l retroviral vector. In a p,crelcd Pmho~limPnt, the SD and SA sites will be sepa,~led by a short, i.e., less than 400 mlclP~oti~e, intron sequP,nce. Such seq~lPncP~s may serve to st~hili7~ RNA LlallSC~ S. Such st~bili7ing seq~lPncPe typically c~....l.. ;ee a SD-intron-SA confi~-ration located 5' to the gene of interest.
15The .cco..... l~ cL~u~ l vectors ofthe invention will also p.crc,~bly contain transcriptional plOlllOLCl~ derived from the gag region opera-hly poeiti~np~l such that a rP~elllt5mt~ sclip~coll~ jing the gene of interest further c~ mprie~Ps a 5' gag I lTR
(ullll~ ted region) u~Llc~l~ of the gene of interest.
The present invention also provides for multivalent vector constructs, the 20 construction of which may require two promoters when two proteins are being ~_AlJl esscd, because one promoter may not ensure ~leq~l~tP, levels of gene e~less;on ofthe second gene. In particular, where the vector construct e,~-c~ es an ~ntiepnee ,..Pe~e ûr ribozyme, a second plullloLcl may not be nPc~e~ . Within certain emborlim~onte~ an internal ribosome binding site (IRBS) or herpes simplex virus thymidine kinase (HSVI~) 25 promoter is placed in c~ l;on with the second gene of interest in order to boost the levels of gene cA~les~ion ofthe second gene. Briefly, with respect to Il~BS, the U1JSL1CaI11 ullLl~ l ed region of the immllnr globulin heavy chain binding protein has been shown to support the internal Png~mPnt of a bicistronic mpee~ge (Jacejak et al., Nature 353:90, 1991). This sequPnre is small, al~pro,~;...~lPly 300 base pairs, and may readily be 30 incorporated into a vector in order to express mllltirle genes from a multi-cistronic mPes~
whose cistrons begin with this sequ~nce Retroviral vector constructs accord.ng to the invention will ofcen be encoded on a pl~emi~l, a nucleic acid mr lecllle capable of propogation, segre~tioni and extrachromosom~ nr,e upon introduction into a host cell. As those in the art will 35 understand, any of a wide range of PYietins~ or new pl~emitle can be used in the practice of the invention. Such pl~emi~le contain an origin of replication and typically are modified to contain a one or mûre mllltiple cloning sites to f~ t~te lecol,-bi~ L use. Preferably, W O 96/33282 PCTrUS96/OS638 pl~em;tlS used in acco~ ce with the present invention will be capable of propogation in both eukaryotic and prokaryoric host cells.

Generation of P~c~in~ Cells - Another aspect ofthe invention relates to mPtho~e of producing ~
tlopic lt;llu~ l partlcles L,cc,l~o~ g 11,_ ~l-uvira~tvectors des~i-il,cd herem. ~n one embo~1imPnt~ vectors are p~Cl~Epd into infectio~s virions through the use of a p~A~n~
cell. Briefly, a p~ inE cell is a cell comprising, in ~ ition to its natural genetic 10 comp'A-nPnt ~d~lition~l nucleic acids coding for those retroviral structural polypeptides required to p~ e a l~I uvL~I ~PnnmP, be it lecr....hi~ (i.e., a rel-ovi,~l vector) or otherwise. The retroviral particles are made in p~rl~n~ cells by ccs~ L~ing the It;llOVil~
ge.~o...P, with a capsid and envelope to make a tr~ned~ction co~.pel~ plert; ~lyreplir~tirn dere~ilive, virion. Briefiy, these and other p<~r1r,A. inE cells will contain one, and 15 preferably two or more nucleic acid mole ~hPe coding for the various polypP,pti~lp$~ e.g, gag, POI~ and env, lt;4uil~d to p~r~ e a retroviral vector into an infectio-le virion. Upon introduction of a nucleic acid mol,A;c -le coding for the retrovira1 vector, the p~r~ n~ cells will produce infectio~s retroviral particles. p~rl~ inE cell lines l ed with a ~ vil~l vector according to the invention which produce infPctiol~c virions are lt:re -t;d to as 20 "producer" cell lines.
A wide variety of animal cells may be utilized to p. ~ e the parlr~in~ cells of the present invention, inr.~ lin~ without limit~ti~n, epithpli~l cells, ~roblasts, hPp~tocytes, endothelial cells, myoblasts, &L..,~,~tes, lymphocytes, e~c.. Plt;r~;lenlially, cell lines are sPlected that lack genom;c sequences which are homf)logo~e to the retroviral vector 25 construct, gag/pol cA~Iession c~es~Ptte and env ~A~res~ion r~eSP~e to be tili7PA MPth~e for deL~ homology may be readily accompli~hpd by, for example, hybri~li7~tion analysis ~Martin et al., Proc. Natl. Acad. Sci., USA, vol. 78:4892-96, 1981; and U.S.S.N.
07/800,921, supra).
The most common parl~ in~ cell lines (PCLs) used for MoMLV vector systems (psi2,PA12, PA317) are derived from murine cell lines. However, murine cell lines are typically not the p~c;re:~ed choice to produce retroviral vectors intPnded for human therapeutic use because such cell lines are known to: contain endogenous retroviruses, some of which are closely related in seq~Pnr,e and retroviral type to the MLV vector system ple~-.t;d for use in pr~ctiring the present invention; contain non-retroviral or defective retroviral seq~lpnres that are known to pac~e effiripntly; and cause deleterious effects due to the presence of murine cell membrane components.
An impo,~-l con~idPration in developing pacl~ging cell lines useful in the invention is the prod~lcti~n therc:~i Olll of replication incompetent virions, or avoi~nre of generating CA 022l687l l997-lO-l7 W096/33282 PCTrUS96/05638 -12-replir~tion-co~p~ retrovirus (RCR) (Mllnrh~-l ef al., Virology, vol. 176:262-65, l99l).
This will ensure that inf~ctiollc ~ ,ov..~l particles harboring the reco~ h;~ .eL,-~v--~l vectors of the invention will be inc~r?blc of independent rerlir~tinn in target cells, be they in vifro or in vivo. Independent replir~tinn~ should it occur, may lead to the pro~h~ctinn of S wild-type virus, which in turn could lead to mllltiple i.,~cg.~.~ions into the chromnsom~(s) of a patient's cells, thereby increasing the possibility of insertional mllt~ n~cic and its zrCsori~ed problems. RCR pr~ cti~n c~r occur in at }east two w~s~
homologous l~co.~ lion between the the.~eulic proviral DNA and the DNA enro~
the le~ vi~l structural genes (ngag/por and "envn) present in the parl~gin~ cell line; and 10 (2) generation of replir~tinn-cQ...pe~e..l virus by homologous ,~o~ ;nn ofthe proviral DNA with the very large ~-u~l~bel of defective endogenous proviruses found in murine parl~gin~ cell lines.
To circumvent inherent safety problems ~csori~ted with the use of murine based ~c;U~ )h~alll retroviruses, as are p-ert;..ed in the practice ofthis invention, par~ng cell 15 lines may be derived from various non-murine cell lines. These include cell lines from various ~..~.-----~lc, in~ in~ hllm~nc dogs, monkeys, mink h~.~le.:j, and rats. As those in the art will app-ec;ale, a mllhitllde of p~r~ng cell lines can be g~le-~led using te~ lu~c known in the art (for i..~ "r.f~, see U.S.S.N. 08/156,789 and U.S.S.N. 08/136,739). In p~c;rt; -~d emhotlim~ontc~ cell lines are derived from canine or human cell lines, which are 20 known to lack g~nomic sequences homologous to that of MoMLV by hybritli7~tionanalysis (Martin ef al., supra). A particularly p-c;r~ed parent dog cell line is D17 (A.T.C.C. ~ccescion no. CRL 8543). HT-1080 (AT.C.C. accrs~;on no. CCL 121; Graham ef al., Vir., vol. 52:456, 1973) and 293 cells ~Felgner et al., Proc. Nat'l. Acad. Sci. USA
84:7413, 1987) lepl. sell~ particularly plt re~t d parental human cell lines. Construction of 25 p~rlrA~ing cell lines from these cell lines for use in conj~lnr-tiQn with a MoMLV based . co..lbialll retroviral vector is decrrihed in detail in U.S.S.N. 08/156,789, supra.
Thus, a desirable pre.t:.luisite for the use of retroviruses in gene therapy is the availability of.. ~....vil~l p~rlrAgin~ cell lines incapable of producing replic~tisn competent, or "wild-type,H virus. As p~cl~A~ing cell lines contain one or more nucleic acid molecules 30 coding for the structural proteins fe4ui~t d to Aespmhle the retroviral vector into infectioll$
retroviral particles, recol~.hinAI;~n events between these various constructs might produce replir~tion Co~ ,tt_~ virus, i.e., infectirJ~ls retroviral particles C~IA;~ g a ~nome encollinp all ofthe structural genes and regulatory Pl~mrnte, inri~ in~ a p~rlrA~in~ signal, required for independent replication. In the past several years, many di~ L constructions 35 have been developed in an attempt to obviate this concern. Such constructions inrl~ e:
deletions in the 3' LTR and portions of the 5' LTR (see, Miller and Buttimore, Mol. Cen Biol., vol. 6:2895-2902, 1986), where two reco.,lb.nalion events are n~ocessA.y to form RCR; use of comrlom~-ntAry portions of helper virus, divided among two sepal ~e pl~emi~le, CA 022l687l l997-lO-l7 W 096/33282 PCTrUS96/05638 one co..~ g gag and pol, and the other co.~ g env (~e, M~l~uwiL;~ et al., J. Virol., wl. 62:1120-1124, and M~uwiL~etal., Vfrology, vol 167: 600-606, 1988), wherethree ~cc....h;~ n events are required to generate RCR
The ability to express gag/pol and env fi-nrtinn separately allows for 5 m~nip~ tiQn ofthese fi~n~ n~ inr1eprnrlP!ntly. A cell line that eAp~ ses ample ~ of gag/pol can be used, for ~ p!c, to address question~ of titre with regard to env. One factor r~ tm~ in ll~ U~1 low titres istke ~tyof ~ ù~ L~ 1~cc;eJLul molecules on the target cell or tissue. A second factor is the affinity ofthe lcc~Lor for the l ~;L uv~l xenotropic envelope protein. One report su~estc that AenuL~upic vector, in the l,-ese-lce 10 of replir~tion-comrl~m~nt xenotropic virus, may more ~Lrt;~iLively infect human hematopoietic progenitor cells (Eglitis, etal., Biochem. Biophys. Res. Comm. 151:201-206, 1988). Xenotropic vector-co. .l ~ .g particles, in the pl esence of repli~tion-cQ...~ .l xenotropic virus, also infect cells from other species which are not easily infPc.t~hlP by amphoL-opic virus such as bovine, porcine, and equine cells (Delouis, et al., 1~ Biochem. Biophys Res. Comm. 169:80-14, 1990). In a pl~;r~llcd emhoflim~nt ofthe invention, p~ 7ng cell lines which express a ~ellolll:)pic env gene are provided.
.Cignifir~ntly, .~:c~ l retroviral partides produced from such p~r1~r~ cell lines are ,S~ I;Ally free from ~ori~tion with replic~tion co...l~e~ relluvi--ls ("RCR").
More l~;cellLly, further improved mrthofls and comrositinn~ for inh;hitin~ the 20 production of rerlir~tion i..co,.-pelenl retrovirus have been developed. See co-owned U.S.S.N. 09/028,126, filed Se~lGllll~Gl 7, 1994. Briefly, the spread of replir,~tiQn competPnt retrovirus generated through lGco~ ;on events between the l~;;CQ~h;~ l rchuvi~
vector and one or more of the nucleic acid con~l. u.;l:~ coding for the ~ ~l- uvi~l structural p.ulci--s may be prevented by providing vectors which encode a non-biologically active 25 il~ ibiLci~y molecule, but which produce a nucleic acid molecllle rnro~lin~ a biologically active inl~ o-y molec~lle in the event of such 1 Gcol--binaLion. The c,~- G~ion of the inl~iLo.y mqlec~lle prevents production of RCR either by killing the producer cell(s) in which that event occurred or by :~ùppl essi Ig production of the retroviral vectors therein. A
variety of inl ,~h;l o. y m~lleclll.os may be used, inr.~ ling riboymes, which cleave the RNA
30 Ll~llscli~l ofthe replic~tiorl co.llpcLG..I virus, or a toxin such as ricin A, tet~n~ or diphtheria toxin" herpes thymidine kinase, etc. As those in the art will appreciate, the te~hings therein may be readily adapted to the present invention.
In ~dl1ition to issues of safety, the choice of host cell line for the p~cL-~ging cell line is of importance because many of the biological properties (such as titer) and physical 35 properties (such as stability) of retroviral particles are ~lict~ted by the properties ofthe host cell. For ;..~ " the host cell must ~ffir;~ntly express (L.~ls-,.ibe) the vector RNA
~nnmP., prime the vector for first strand synthesis with a cellular tRNA, tolerate and covalently modify the MLV structural ploteills (proteolysis, glycosylation, myristylation, W 096/33282 PCTrUS96105638 and phosphnrylation), and enable virion budding from the cell ~--en.l,-~le. For ~ pl~, it has been found that vector made from the mouse par~nS~ line PA317 is retained by a 0.3 micron filter, while that made from a CA line will pass through. FU.LI.C....OIC~ sera from p.il..ales, inrlurlin~ hllm~ne but not that from a wide variety of lower ,..h.."..~le or birds, is 5 known to inactivate retroviruses by an antibody independent compl~~Pnt Iysis mPtho~l Such activity is non-selective for a variety of di:iL~I~ly related retroviruses. Rc~ Vil uses of avian, murine (inrlt~inS~ MoMLV), feline, and s~m~an ongm are inactlvate~and ryse~by normal human serum. See Welsh ef al., (1975) Nature, vol. 257:612-614; Welsh et al., (1976) Virolo~y, vol. 74:432-440; Banapoul ef al., (1986) Virology, vol. 152:268-271; and 10 Cooper el al., (1986) Immunology of t*e C~ nt System, Pub. ~mPrir~n Press, Inc., pp:l39-162. In ~cl~1itirn replir,~tiQn Co~pe~ murine ~mphntropic retroviruses il~je~le~
vc;nously into p.il..a~es in vivo are cleared wit-hin 15 ~~ es by a process ...P~ pA in whole or in part by primate cc ~..plrmPnt (Cornetta et al. (1990), Hz~man Gene T*erapy, vol. 1:15-30; Cornp~tt~ etal. (1991), Human Gene T*erapy, vol. 2:5-14). However, it has lece.llly been discov~;led that retroviral reeiet~nre to comp'~ nPnt inactivation by human serum is ~-.~ le~l, at least in some i,~ e, by the p?~rl'~ ing cell line from which the retroviral particles were procl~lce~ Retroviruses produced from various human p~:L :~gJng cell lines were resistant to inactivation by a colllpone -~ of human serum, p-e~compl~ nPnt, but were sensitive to serum from baboons and m~cques See commonly owned U.S.S.N. 081367,071, filed on DecPmhPr 30, 1994. Thus, in a plere ~ed embodiment ofthe invention, reco...hil-h..l retroviral particles coding for full length factor VIII are produced in human p~ g~ng cell lines, with p~r.lr~ng cell lines derived from HT1080 or 293 cells being particularly p-er~;llt;d.
In ~cl~itinn to genel~-Lil~g infectirJIle rep!ir~tion defective l~co...bi~ retroviruses 25 as described above, at least two other alternative systems can be used to produce ,t;co...l-i.~..l retroviruses carrying the vector construct. One such system (Webb, et al., BBRC, 190:536, 1993) employs the insect virus, baculovirus, while the other takes advantage of the " "." "~ n viruses vaccinia and adenovirus (Pa~nl ~1, et al., BBRC, 145:234, 1987). Each of these systems can make large amounts of any given protein for 30 which the gene has been cloned. For PY~mplP., see Srnith, ef al. (Mol. Cen Biol., 3:12, 1983); Piccini, et al. (Meth Enymolo~, 153:545, 1987); and M~nsol-r et al. (Proc. Natl.
Acad. Sci. USA, 82:1359, 1985). These viral vectors can be used to produce ploLeil~s in tissue culture cells by insertion of applol,.iale genes and, hence, could be ~ pte~ to make retroviral vector particles from tissue culture. In an adenovirus system, genes can be 35 inserted into vectors and used to express proteins in ..-~ n cells either by in vitro construction (Ballay, ef al., 4:3861, 1985) or by reco,llbillalion in cells (Thllm mPl, et al., J. Mol. AppL Genefics, 1 :435, 1982).

CA 022l687l l997-lO-l7 WO 96/33282 PCTrUS96/05638 An al~ ,aliv~ approach involves cell-free p~ gin~ systems. For i~ r~;
retroviral structural prole.ns can be made in a baculovirus system (or other protein production ~ s, such as yeast or E. coli) in a similar manner as descrihed in Smith et al.
(supra). Rec~ bil~a,.l retroviral ~nn~ -s are made by in vi ro RNA synthesis (see, for ; . '~, Flamant and Sorge, J. Virol., 62:1827, 1988). The structurat p-olei-,s and RNA
g~nnmrs are then mixed with tRNA, followed by the ?~d~lisit~n of liposQmr~ with Pmhedded c~v ~ ,lein and cell extracts (typically from mo~lse cells~ or y~ J c~ rl~Ol~ (which provide env and other nrcç~ y procec~ing and any or other nece~ cell-derived fi-nr,ti~n~). The ll~iAIu.ei is then treated (e.g, by sonic~ti~n~ te",ye,~LIu~ vl~l;Qn~ or rotary dialysis) to allow Pnc~r~ ti~n of n~crnt rt;l.ùvi,~l particles. This procedure allows producti~n of high titer, replir~ti~n i~-co~ eco~ el~ùvh~lses without c s.-l~ n with pathogenic retroviruses or rep1ir,~tir~n-cQ~ e~ l retroviruses.
Another i~yO~ factor to con~;~lrr in the selection of a p?~cl~n~ cell line is the -viral titer produced thereL u", following intro~ cti~n of a nucleic acid molecllle from which the retroviral vector is prod~lce-i Many factors can limit viral titer. One ofthe most si~ifir,~nt lirniting factors is the ~yres~ion level of the p~rl~n~ proleins gag, pol, and env. In the case of retroviral particles, ~ yres:iion of retroviral vector RNA from the y,ùv"us can also cigrlifir~ntly limit titer. In order to select p~ins~ cells and the rPsult~nt producer cells c~.ylessi~g high levels ofthe re4u,led products, an app,ùy~iaLe titering assay is required. As described in greater detail below, a s ~it~ble PCR-based titering assay can be ~ltili7P,~l In ~ddition to prèpa~ g p?~rl-~gin~ and producer cell lines which supply p,uleills for p~r.lr~gin~ that are h- mt~l~ollc for the h~cl~honp of the viral vector, e.g, I el, ùv" ~I gag, pol, and env proteins for p~r~ 1nF~ of a retroviral vector, p~rl~s~ in~ and producer ~:ile~S
which result in chimeric viral particles, for i~cl~i~ce a MoMLV-based retroviral vector p~cl~z~ed in a DNA virus capsid, may also be employed. Many other p~cl~gin~ and producer ~y~le",s based on viruses u~el~led to that ofthe viral vector can a1so be lltili7e~
as those in the art will apy, eciale.

Altering the Host Range of Reco~bi~a~ll Retroviral Particles Another aspect of the invention concerns reco~ l xenotropic retroviral particles which have an altered host range as co",p~ed to retroviral particles CQ~
arnphotropic envelope proteills~ The host cell range sperifirity of a retrovirus is detprmin~d in part by the env gene products present in the lipid envelope. L,lele~ gly, envelope proteins from one retrovirus can often s~lbstihlt~p~ to vary-ing degrees, for that of another retrovirus, Ihelel~ altering host range ofthe rPelllt~nt vector. Thus, p~clr~ging cell lines (PCLs) have been genel~Led to express either arnphotropic, ecollùpic, xenotropic, CA 022l687l l997-lO-l7 W 096133282 PCTrUS96/05638 polytropic, or other envelope l.ui).sl.,s. ~ tion~lly, retroviruses acco-d-ng to the invention which contain "hybrid" or "ctlim~ric" xenotropic envelope proleil~s can be similarly genc.~led. Retroviral particles produced from any-ofthese pa~l~nf~ cell lines can be used to infect any cell which cQ..Ini~Q the cQ--e~on~ g distinct receplor (Rein and Sch~llt7:, Virolo~, 136:144,1984).
The ~QQPmhly of-Gl.ovil~ses is char~ .;,ed by selective in~ Q;on ofthe rclluvi.~l gPnome and ACC~ O-y p. uleil~s into a ~u~.~ ,vil~ ..h. L,~ 3~, c,.~,.,h~pc~
proteins from non-murine retrovirus sources can be used for pse~ldotyping (i.e., the en~rQ;d~tion of viral RNA from one species by viral prùleins of another species) a vector lû to alter its host range. Re~ ~e a piece of cell ll-e ~-lJ~Ie buds offto form the IGI-uv~l envelope, molec~ c normally in the me...l,.a.le may be carried along on the viral envelope.
Thus, a number of difrerG~I pulG--lial ligands can be put on the surface of ~GL~vh~l particles by m~nir~ tinf~ the p~ Eing cell line in which the vectors are produced or by choosing various types of cell lines with particular surface .--~I,c ~.
1~ Briefiy, in this aspect the present invention provides for enveloped lt:llOVilil]
particles co-~ a ml~leoc~rQ-id in~ lir~ mlcleoc~rs;d protein having an origin from a first virus, which is a lelluvi~ , a p~cl~ge~ble nucleic acid mnlecllle Pn~otlinf~ a gene of interest ~Q-Q-o~ ted with the nll~ leo~rs;~l; and a ~..e.l~l~le-~QQori~tPA xenotropic protein which d~ es a host range.
In another plcrelled form ofthe present invention, the .llcll~ e-~e~ori~te~d envelope protein of the vector particles is a chimeric or hybrid protein inr~-lrlin~ an exterior receptor binding domain and a l--Gl--I,.~e-~esori~ted domain from a Aenol~or c envelope protein, at least a portion of the exterior receptor binding domain being derived from a diLrc-cnl origin than at least a portion ofthe membrane-~esori~ted dom~in The chimPric protein is preferably derived from two origins, wllGIGill no more than one ofthe two origins is retroviral.
Another embodiment of this aspect of the present invention conr~ .e cell lines that produce the foregoing vector particles. Preferably, such cell lines are stably ll;...~rr~ed with a nucleic acid moleclllp~ Pncorlin~ the membrane-~eeori~tPd protein, whose ~,A~.Iession 30 is driven by an inducible p.u,..olel.
Retroviral particles according to the invention may be targeted to a specific cell type by inrl~ltling in the retroviral particles a component, most frequently a polypeptide or carbohydrate, which binds to a cell surface lt;ce~lor specific for that cell type. Such targeting may be ~ccQmrliehPd by p-ep~i--g a p~rl~gin~ cell line which cAylesses a 35 chimeric env protein collll~lismg a portion ofthe env protein required for viral particle assembly in conj~nr,tinn with a cell-specific binding domain. In another embo~lim~nt~ env proleins from more than one viral type may be employed, such that res~lt~nt viral particles contain more than one species of env proteins. Yet another embodiment involves inr~ )n CA 022l687l l997-lO-l7 W 096/33282 PCTrUS96/OS638 -17-of a cell specific ligand in the retroviral capsid or envelope to provide target ~ererifirity. In a pLerel-cd embo~im~nt at this aspect ofthe invention, the e~v gene ~ ~ ncodes all or a portion ofthe env protein le~lui ed for retroviral asse.~-bly in conj~1nrtion with a ~ Icce~lor binding domain of a polypeptide ligand known to interact with a cell surface 5 receplor whose tissue ~ .onis limited to the cell type(s) to be ~cled, e.g, a T cell.
In this regard, it may be prcrcl~'e to utilize a leceplor binding domain which binds cplol~ CA~ .sed at~r levels on th~t~rget ~ssur~.
In order to control the specific site of in~eg-~lion into a patient's ~n~m~ in those r~e where the vector construct employed leads to integration of the viral g~nomç into 10 a chr~ mosome ofthe rerip:~- t cell, as occurs in the case of .cl,uvu~ infer,tinn homologous reco...1~ -l ;on or use of a m~ if iPd integrase en_yme which directs insertion to a specific site can be utili7er~ Approaches for the use of integrase pl oleins to direct site specific u-~eg.~ion is described in WO 9l/02805 entitled "p~eco~h;~A~ Re~rovuuses Delivering Vector Constructs to Target Cells" and co-owned U.S. app1ic~tior~ No. 445, 466, filed May 22,l995, both of which are hereby incorporated by rcreltl-cc. Such site-specific insertion of the vector c~lyulg the gene of interest may provide for gene reE~1~c~m~nt therapy, reduced c1-A~u~s of insertional mllt~g~n~cie~ ...;.~;...;,.~ i..lelrclc"cc from other sç~ nce5 present in the patient's DNA, and allow insel Lol- at specific target sites to reduce or cl;~ A~ e cA~I cssion of an undesirable gene (such as a viral or tumorigenic gene) in the patient's DNA.
Non-viral ~,I~,--I,.~e-~cso~ ted pluLcills may a1so be used to e~1-A~-,e l~;cli"g of Icco-~h;l~h~-l IcLluvu;ll particles, in~ g xenotrophic 1CIlUVil;:~1 particles, to T cells.
Rep, cse~llaLive c r 1cs include polypeptides which act as ligands for T cell surface receptors. Depclld.l~g on the tissue distribution ofthe .~cel,~or for the protein in ~1eletion the Icco"-l~u~u~l Aenol.opic lcll~,vudl particle could be ~c~ed to a diLre c"t subset of T
cells.
When a ligand to be in- 11lded within the envelope is not a naturally oc.;~ ~ . ;.-g me",h,~le-~eeoci~ted protein, it is ~ecçssh~y to ~ecori~e the ligand with the me...hl~c, p~crcl~bly by making a "hybrid" or "chimeric" envelope protein. It is i,..po.~.l to 30 understand that such hybrid envelope proteins can contain extr~cP11~ r domains from proteins other than other viral or retroviral env proteins. To ~ccomplish this, the gene coding for the ligand can be functionally comhined with sequences coding for a ~--cn-h~ e-associated domain ofthe env protein. By "naturally occurring membrane ~ceori~tedprotein", it is meant those proteins that in their native state exist in vivo in ~eeo~ i~tir)n with 35 lipid membrane such as that found ~ceo~i~ted with a cell me~ hlane or on a viral envelope.
As such, hybrid envelopes can be used to tailor the tropism (and effectively increase titers) of a retroviral vector according to the invention, as the extr~c~ r component of env proteins is responsible for specific receptor hint1ing The cytoplasmic domain of these W096/33282 PCT~US96/05638 ploleins~ on the other hand, play a role in virion form~tion The present invention recognizes that l-u---e-uus hybrid env gene products (i.e., sperifir~lly~eLIuv~ env proteins having ~,yLoplaslllic regions and extracP~ lAr binding regions which do not naturally occur together) can be genel~led and may alter host range specificity.S In a plère~ d embo~lim~nt this is accQn rliehp!d by l~c~.. l~ g the gene coding for the ligand (or part thereof ccjllrelling ~ece~llor binding activity) p,u~ e ofthe l"e.~ e-bindingdomain ofthe envelope proleills that staJo}~ g~Rr capS~ plvlw-l.
The rçsnlting construct will code for a bifimr,tionA1 ~I.;.--~. ;c protein capable of fl~hAI~red cell la~gèLi~lg and inrl.l~;on in a rèLluvi~l lipid envelope.
Vector particles having non-native lllelll~ e-Ae-eor;~t~ ligands as sl~e~ - ;hedherein, will, advAnt~oQusly~ have a host range dele~ d by the ligand-rèceplor interaction ofthe ~~e~lbl~le-Aeeor;~ted protein. Thus, for targeted delivery to T cells, a vector particle having altered host range can be produced using the mPthods ofthe present invention. The ligand will be se1ected to provide a host range inr~lutli~ T cells. Many diLrere~L l~ugeling strategies can be employed in conn~ction with this aspect ofthe mvent~on.
Antibodies may be also utilized to target a s~lected cell type, such as anti-CD4antibodies to target CD4+ T-cells and anti-CD8 antibodies to target CD8+ cells (see generally, Wilchek, etaL,Anal. Biochem., 171:1, 1988).
T Iymphocytes or T cells are non-antibody produring Iymphocytes that col.e~ lethe part of the cell-~ Pd arm of the immlme system. T cells arise from ;.. ~ eIymphocytes that migrate from the bone nl~luw to the thymus, where they undergo a ... ~lu.~lion process under the direction ofthymic h~rmcmes Here, the ;.. ;~ e lymphocytes rapidly divide increasing to enormous ~.u---I)e ~. The ...~m. ;..~ T cells become 25 immlml~CC~ by having the ability to recogni7e and bind a specific ~ntig~n Activation of ~ competent T Cells is triggered by antigen binding to the Iymphocyte's surface receplol~.
T cells can be isolated by a variety of procedures known to those skilled in the art.
For eY~mrl~ crude T cell suspensions can be prepared ~om spleen and Iymph nodes by 30 passing homogenates through nylon wool columns (Current Protocols in Tmmlmolnpy, Coligan, et. al. (1992) Green Publishing ~soci~tes and Wiley-Intersrienr~ New York).
This procedure offers a convenient means of l~.nri~hinp~ T cell poplll~tion~ through the removal of accessory and B cells. T cells from mouse spleen and lymph node do not express the cell-surface gly~,ol)Loleins en~ o~led for by MHC class II genes, whereas most W O 96/33282 PCTrUS96/05638 non-T cells do. The,~ro~e, T cell enrichmP-nt can be ~ccomrlichPd by the ç~ ;on of non-T cells using anti-MHC class II m~nsClonAl Antibo~liPs Similarly, other antibodies could be used to deplete specific pop~ tiQn~ of non-T cells. For Py~mple~ a-Ig for B cells and a-MacI for macrophages.
T cells can be further fMctir~n~ted into a number of diLrelenl subpoplll~tinn~ by tect~ l.,es known to those skilled in the art. Two major sul,p.3p~ I;o.~ can be i~nl~tPd based on their diLrer~llLal eA~.ession ofthe cell surface ~ kel:icD4 and CD8. For P,YAmrlP following the çnrirhmpnt of T cells as described above, CD4+ cells can be Pnrichp~d through the use of antibodies specific for CD8(d~psrribed in Current Protocols in 0 Tm mlm-logy,supra ). ~ltprn~tively~cD4+ cells can be enriched through the use of antibodies specific to CD4, cQ~1pled to a solid ~u~)pGIl such as .~Ag~ ,l;r, beads. Conversely, CD8+ cells can be enriched through the use of antibodies specific for CD4, or can be ic~lAted by the use of CD8 antibodies collrled to a solid support. CD4 Iymphocytes from HlV-l i-~re~iled p~tientc can be PYr~n(1ed ex vivo, before or af'cer k~n~duction~ as des~,il,ed by Wilson et. ~. (J. Infect Dis172:88,1995).
Following p~lrifir~tion of T cells, a variety of mPth~-ds of trAncductinn known to those skilled in the art can be l,c,rul",ed. For PY~mple~ one such approach involves trAn~dllrtion ofthe purified T cell poplllAfiQn with vector cc,..l~ r~e~ .1 cultures derived from vector producing cells. A second approach involves co-cultivation of an irradiated monolayer of vector produri~ cells with the purified T cells. A third approach involves a similar co-cultivation approach, however the pllrified T cells are pre-stimlll~tçd with various cytokines and ~;ulluled48 hours prior to the co-cultivation with the irradiated vector produring cells. Pre~ ;on prior to trAn~duction increases effective gene l~,srer ~Nolta et al., Exp.~PmAtol20:1065;1992). While not wishing to be bound by theory, the increased level of trAn~ductir n is attributed to increased proliferation of the T
cells ~ce~sA~y for çfflri~ont retroviral tr~n~duction Stim~ tinn ofthese cultures to proliferate also provides increased cell pop~ tiQn~ for re-infusion into the patient.
Subsequent to co-culliv~lion, T cells are collected from the vector produrin~ cell monolayer, ~ypAntled~ and frozen in liquid nitrogen. The c~c~,,ion of vector in tr~n~ r,ed 30 cells can be ~cs~i~ed by a number of assays known to those skilled in the art. For ,; , Iç, Western blot or Northern analysis can be employed depending on the nature of the inserted gene of interest. Once c A~l es.,ion has been established and the transformed T cells have W096/33282 PCTrUS96/05638 been tested for the p, c;sence of advP-ntitious agents, they are infused back into the patient via the pe~iph~l~l blood stream.
Those in the art will also rec~i7~ that it is also po,eQ ~'~ to add ligand m~ cllle~
exoge~ to the lt;h~)vil~l particles which are either L.lcol~ol~ed into the lipid envelope 5 or which can be linked ehPmir~lly to the lipid or protein cor. ~ Pnte thereo~
In ~Itlition, a wide vanety of high affinity binding pairs can ~e usea' as k.l ~u~
Repl~ek~ v~ s of include biotin/avidin with an affinity (KD) of 10-l5 M
~Richards, Meth. Enz., 184:3, 1990, Green, Adv. in Protein Chem., 29:85, 1985) and cystatin/papain with an affinity of 10-14 M ~Bjork et al., Riocl~k~ y, 29: 1770, 1990). A
10 wide variety of other high affinity binding pairs may also be developed, for; . 'e, by pl~illg and sPlecting ~ntiho~ip~s which recognize a srlo~led T cell antigen with high ~ffinity (see generally, U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993;
see also Monoclonal Antibodies, Hybritlom~e A New D;...~ Ol~ in Biological Analyses, Plenum Press, KPnnPtt McKP~rn, and RerhtQl~ eds., 1980, and Antibodies: A Labo~
15 l~ml~l Harlow and Lane eds., Cold Spring Harbor Laboratory Press, 1988). The binding pair for such antibodies, typically other antibodies or antibody L;., ...~ may be produced by recc,llllfill~lL techniques (see Huse, et al., .Sr;enrP; 246:1275, 1989; see also Sastry, et al., Proc. Natl. Acad. Sci. USA, 86:5728, 1989; and MirhPllP, Alting-Mees, et al., StrategiesinMolcc~ rBiology, 3:1, 1990).
As will be evident to one of ol~ skill in the art given the ~lierlosllre provided herein, either melnber (or moleclllp~) ofthe affinity binding pair may be collplc~l to the retroviral particle. Nevertheless, within pl er~ d embodiments of the invention, the larger of the two affinity binding pairs (e.g, avidin of the avidin/biotin pair) is co~lple~l to the lt;LI~vilal partide. As utilized within the context of L2~;t;Lih~g, the term "collplcd" may refer to either noncovalent or covalent interactions, although generally covalent bonds are plt:r~llt;d. Numerous coupling metho~le may be utili7p~ inrl~l-ling for PY~mrlP; use of cros~linkin~ agents such as N-s~lcrinimidyl-3-(2-pyridyl dithio) propionate ("SPDP";
Carlson, ef al., J. Biochem., 173:723, 1978) and other such compounds known in the art.
In particularly pler~;llt:d embodiments ofthe invention, a mPmhPr ofthe high affinity binding pair is either eA~,lessed on, or inrlllded as an integral part of, a retroviral particle, e.g, in the retroviral lipid envelope. For ~ .ple, a member ofthe hig~h~ affinity binding W 096/33282 PCTrUS96/05638 pair may be co eA~ ssed with the envelope protein as a hybrid protein or ~ e~sed from an app-op,iaLe vector which targets the mPmhPr of the high affinity binding pair to the cell l"~",b,~le in the proper ori~Pnt~tinn 5 Uses of Reco~ Retroviral Pal l;~ les In one aspect, the present invention provides mPth~c for inh;1~itin~ the growth of a selected tumor ("cancerN) in an human, comprising the step oftr~nc(l~lci~ T cells ex vivo with a vector construct which directs the ~ ~ression of at least one anti-tumor agent.
10 Within the context of the present invention, "inl~ili,lg the growth of a sPlected tumor"
refers to either (1) the direct inhi~ition oftumor cell division, or (2) ;---------lç cell m~i~t tumor cell lysis, or both, which leads to a :j~ples:jion in the net I l-AI~c;on oftumor cells.
Tnhil~itisn of tumor growth by either of these two .~pch~ c may be readily d~l~- ...;-.P~1 by one of o, di"~y skill in the art based upon a "uml~er of well known mçthsrlc for 15 1 -; ~-~ by mP~cllring the tumor si_e over time, such as by radiologic i~.A~p mPthnrlC
(e.g, single photon and positron çmicQ;~n co",l,u~ ed tomography; see generally,~NUdear MPt1;r;nP-1n Clinical Oncology," Winlcler, C. (ed.) Springer-Verlag, New York 1986)orbyavarietyof ;...Agil~g agents,in~ in~ for; le,convPntion~l im~n~
agents (e.g, ~ m_67 citrate) or spec;~li7Pd re_gellls for mpt~bolite ;-..~g;i~P receplor 20 im~n~, or immllnologic im~gir~ In a~ itisn~ non-r~(lio~ctive mPthsds such as ultrasound (see, "Ultrasonic Di~ere"L,al Di~nssic of Tumors", K9scoff and Fukuda, (eds.), Igaku-Shoin, New York 1984), may also be utilized to P~;l;-..~e tumor size. ~ltprn~tively~ for other forms of cancer, inhibition of tumor growth may be d~le""illed based upon a change in the presence of a tumor marker, e.g, prostate specific antigen (nPSAn) for the detectinn of prostate cancer (see U.S. Patent No. Re. 33,405), and Carcino-Eml"yoilic Antigen ("CEA") for the detecti~n of colorectal and certain breast cancers. For yet other types of cancers such as le~lkPm;~ inhibition oftumor growth may be d~le- ...;I~Pd based upon decreased ~uml)e~ ~ of le -kPmic cells in a representative blood cell count.
Within the context of the present invention, "anti-tumor agent" refers to a 30 co",poulld or molecule which inhibits tumor growth. Rep,t:se~Li~e ~ ..plcs of anti-tumor agents include imm--nç activators and tumor proliferation inhibitors. Briefly, immlmP
activators fim~i~n by improving immllnç recognition of tumor-specific ~nti~Pnc such that W 096/33282 PCTrUS96/05638 -22-the immlme system becomPs ~,lil..ed." Prin~ing may consist of lymphocyte prolirel~lion, dirrèl~ l;on or evolution to higher affinity interactions. The ;.. ~-c system thus primed will more t Lre.;livtl~ inhibit or kill tumor cells. Tmml-nP activation may be ,~ll,ç~l-P~
into immllne mn~ tors (mn~ ps which affect the interaction between lymphocyte and 5 tumor cell) and lymrhokin~s, that act to proliferate, activate, or dirrèlt;lLdle ;~ e ef~ lUl cells. R~lesenl~Live c ~ es of;----..-~-e Inndllr~tQrs inc~uae OEJ~ M-l,ICAM-2, LFA-1, LFA-3, b-2-microF~lobll~ ,I,a~elones, alpha ill~e,relon, gamma .I~Lelrt:lun, B7/BBl and major hietoCQ~ AI;1.;1ity comr" ~IC), and T cell le-~eplor proteins or synthetic equivalents such as T cell receptors with Innrlified reco~i~ion sites.
10 R~resenlaLive Py~mrlos of lymphokin~s include gamma i,~lelrt;lol" tumor ne.;lo~is factor, IL 1, IL-2, IL 3, IL4, IL 5, IL 6, IL 7, IL 8, IL-9, ~10, II,l 1, GM-CSF, CSF-l, and G-CSF. In 7/dt1itinn~ RNA moloclll~5 having intrinei~ biologic-~l activity may be utilized as anti-tumor agents.
Sequ~n~es which encode anti-tumor agents may be ob~ d from a variety 15 of sources. For; , '-, pl~mi~ls that contain sequl~nces which encode anti-tumor agents may be obt~in~d from a depository such as the ~m~ric~n Type Culture Col~ m (ATCC, Rockville, Ma~yland), or from com c;al sources such as British Bio-Technolo y T~imited (Cowley, Oxford F ~ ~n~ tprn~tively~ known cDNA seq~ncPs which encode anti-tumor agents may be obtained from cells which express or contain the seq~PnePS
20 Additionally, cDNA or mRNA libraries from specific cell sources can be pulchased from cc~.. ~cial sources from which the desired seql~Pnees can be readily cloned by collvenlional techni~ e~ e.g, PCR ~mplifi~tion Seqllenc~Ps which encode anti-tumor agents may also be synthPei7er1 for eY~mplP; on an Applied Biosy~ellls Inc. DNA
synthpci7pr(e~g~ ABI DNA synthpci7pr model 392, Foster City, California).
In ~ iti~n to the anti-tumor agents described above, the present invention also provides anti-tumor agents which comprise a fusion protein of, for PY~mple, two or more cytol inP~ immllne modulators, toxins or dirrtle~ ;on factors. ~rerelled anti-tumor agents in this regard include alpha il~Ltlrelull - Interleukin-2, GM-CSF - IL~, GM-CSF -IL-2, GM-CSF - IL-3 (see U.S. Patent Nos. 5,082,927 and 5,108,910), GM-CSF - gamma il,lt,rtlull, and gamma ill~elrelon - IL 1, with gamma i,lLelreroll - TntPrlellkin-2 being particularly pler~;llt:d.

W 096/33282 -23- PCTrUS96/05638 Within another embo~ C, the anti-tumor agent may further ~ a ."~;"Lr;~le anchor The ~--enL.~e anchor may be s~lected from a variety of seq~fenr~q, inrl~ ing for ~ r~ the l~;.n~ e domain of well known p~uLeLs Generally, me ~-I,.~le anchor seql~P-n~f s are regions of a protein that anchor the protein to a ~ 5 ~c~L~le Cl~lo.. ~ ;ly, there are two types of anchor sequonr~s that attach a protein to the outer surface of a cell ,--en~ e~ e regions that span the lipid bilayer ofthe cell ",c;",b,~ e (p-oleins CQ..~ such regions are .~rt;.. d to integral ~e~L~
proleins); and (2) dom~inc which interact with an integral ~--t--~ e protein or with the polar surface ofthe ~--e--L.~e (such prole,..s are referred to as peliphel~l, or ~YtrinQ;~
10 prole--.s) Me...b.~e anchors derived from integral ~--cil.-bl~e prole---s are p-t;r~ ~ed Me.lL.~Ie ~ l1; .g regions typically have a similar structure, with a 20 to 25 amino-acid residue portion co~ P almost entirely of hydrophobic residues located inside them~..L.~Ie (see Fic~h~..g et al, Anm Rev. Bioc~em. 53:595-623, 1984). M~nLl~ e 15 s~ P regions typically have an alpha helical structure (see Ficenl~pig et al supra; Heijne and Manoil at supra) Within a pr~rt;l.t:d embodiment, a ~c~Ll~e anchor is fused to the C-terminus of gamma ~IlLelrt;-oll fusion protein, wherein the ...enL.~ule anchor comprises the gamma-chain ofthe Fc ~~ce~lor Tllmorip~ y of an anti-tumor agent can be ~csfssed by various assays 20 Represf~ e assays include tumor form~tion in nude mice or rats, colony form~tion in soft agar, and p-ep~l-on of lli~Sge~l'C ~nim~lc, such as ~r:~nCg~I~;G mice In ~d~lition to lu~o~el~icity studies, it is generally preferable to d~Le--ll--le the toxicity of an anti-tumor agent A variety of mf tho~ls well known to those of skill in the art may be utilized to measure such toxicity, inrl~lin~ for Py~mplp~ clinical ~-i.f- ..i~l y assays which measure the 25 systemic levels of various prole,--s and enzymes, as well as blood cell volume and number Once an anti-tumor agent has been sfle. Ie~l it is placed into a vector construct acco-d"lg to the invention Such a vector construct can then be packaged into a r ecol.Li"a,.l retroviral vector and be used to tr~ncd~lce ex v~vo T cells which are then re-introduced into the 30 patient In the context of the present invention, it should be understood that the removed cells may not only be returned to the same patient, but may also be utilized to inhibit the growth of sPle-cted tumor cells in another allogeneic human W 096/33282 PCTrUS96/05638 Within one embotlimsnt the r~ ...hi..~ vector construct directs the ~~A~le~ion of a protein or active portion of a protein that binds to newly synthpsi7pd MHC
dass I mn~eclllPc intr~cP~ rly. This binding p~ève,l~s migration ofthe MHC class I
molr,cl-lP~ from the endoplasmic rcticvl~-m, rPe ~ltin~ in the inh;hitisn of terminal 5 glycosylation. This blocks ll~ spo~ ofthese molrJclllps to the cell surface and p~evcllLS cell leco~lilion and Iysis by CTL. For ;.,~ r~, one ofthe ~ U~ ofthe E3 gene maybe used to inhibit transport of MHC class I molec~lss to the surface ofthe l.~lsr~,lmed cell.
More specifically, E3 Pnrodes a 19kD tr~nem~p~hl~le glycopro~eil~, E3/19K, ~ sclil,ed from the E3 region of the adenovirus 2 Eennms Within the context of the present 10 invention, a multivalent recQ...l-;l-~..l viral vector construct is ~tlminiet~pred directly or illdi~e~lly~ and co..l~i..c a gene enco~lin~ a thel~cu~ic protein and the E3/19K sequpn which upon cAlJIes:~ion~ produces the thel~peu~ic protein and the E3/19K protein. The E3/19K protein inhibits the surrace c,~les~ion of MHC class I surface molcrlllPe~ inrl~
those MHC mnl xlllps that have bound peptides of the therapeutic protein. Consequently, 15 cells transformed by the vector evade an ;.--~-....-e responee against the thel~eu~ic protein they produce.
Within another embodiment of the present invention, the multivalent eco. ~ .hi~ . - - .l vector construct directs the e~lJI e~ iOn of a ~hel ~eulic protein and a protein or an active portion of 8 protein capable of binding ~2-microglobulin. Transport of MHC
20 class I molcclllPs to the cell surface for antigen p~ee~ lir~n le~luilès association with 132-microglobulin. Thus, pluleins that bind ,B2-micro~loblllin and inhibit its association with MHC class I indirectly inhibit MHC class I antigen presPnt~tir~n ~l~it~hle proteins include the H301 gene product. Briefly, the H301 gene, obtained from the human ~lu...P~lr~virus (CMV) enr,Qdç~e a glycoproleill with sequence homology to the ~2-microglobulin binding site on the heavy chain ofthe MHC class I mnlp~clll~ (~3rowne et al., Nature 347:770, 1990). H301 binds ~2-microglobulin, thereby preventing the ,.,a~ul~ion of MHC class I
moleclllPe and renders "~ ru ed cells unrecogni7~hle by cytotoxic T-cells, thus evading MHC class I restricted immlmç survp~ n~ e Other proteins, not ~lieclleeed above, that filnction to inhibit or down-regulate MHC class I antigen prPsPnt~tion either generally or more specifi~lly for the specific foreign proteîn Pn~oded may also be i~1Pntified and utilized within the context of the W 096/33282 PCTrUS96/05638 present invention. In order to identify such prole.l~s, in particular those derived from ... ~.. ~li~n pathogens (and, in turn, active portions thereof such as the EBNA-l gly-ala repeat from EBV virus), a recc....l~ vector construct that ~_A~esses a protein or an active portion thereof either as a se~ le entity or fused to the active protein ~ e~iled of 5 being capable of inhibiting MHC class I antigen plesf~ .l ;on is 1, n- ~er~ ç(l into a tester cell line, such as the murine ce}l ~ne BClOME ~see WO 91/02805, entitled "Reco~
Rc~ovil~lses Delivering Vector Constructs to Target Cells"). The tester cell lines with and without the sequence Pnrotling the ç~nt1id~te protein are co---~ cd to stim~ t~rs and/or targets in the CTL assay. A decrease in cell Iysis co"ti;,~onding to the l~n~eru~ ...PA tester 10 cell in~lir~tes that the c-~n~ te protein is capable of inhibiting MHC preS~ n An alternative mPthod to dc~c~ e do~,vn-re~ tion of MHC class I surface cA~ ion is by FACS analysis. More srerifir~lly~ cell lines are l,nn~r.,....çd with a bin~ll vector construct ~nco~ling the c~n~lid~te protein. A~er drug sPlçction and PYr~neion the cells are analyzed by FACS for MHC class I c,~ es:,ion and co-l-p~cd to 15 that of non-l.~.sru,...ed cells. A decrease in cell surface ~,A~ ..sion of MHC class I
inrlir~tçs that the ç~nt~ te protein is capable of;..h~ p MHC pieee~ l;rJI) Any of the gene delivery vehicles described above may inr,lude, contain (and/or express) one or more heterologous seqll~nrPe A wide variety of heterologous sequenr,es may be utilized within the context of the present invention, inrl~ inp for 20 c "!~ lolc,~ic genes, disease-~eQor~tecl ~.I;g~l~Q, _..I;e~ ee seq~l~nr,~c, sequ~n-~Q
which encode gene products that activate a co...~u.l..d with little or no "ylOlOx;~ity (i.e., a "prodrug") into a toxic product, sequences which encode immlmngenic portions of disease-~e.eor.i~ted ~nti~PnQ and seq~lPnc~s which encode ;.",..",.e ~ccPeso-y mnleclllP,s Rep..;;s~ re PY~mpl.o~ of ~yLolo~c genes include the genes which encode proteins such as ricin ~Lamb et al., Eur. J. Biochem. 148:265-270, 1985), abrin (VVood et al., Eur. J.
Biochem. 198:723-732, 1991; Evensen, et al., J. of Biol. Chem. 266:6848-6852, 1991:
CollinsetaL,J. of Biol. Chem. 265:8665-8669, l990;Chenetal.,Fe~ of Eur.Biochem Soc. 309:115-118, 1992), diphtheria toYin (Tweten et aL, J. Biol. Chem. 260:10392-10394, 1985), choleratoxin (~l~nns etal., Nature 306:551-557, 1983; ,S~n~hP7 &
r~olmEren~ PNAS 86:481-485, 1989), gelonin (Stirpe etal., J. Biol. Chem. 255:6947-6953, 1980), pokeweed (Irvin, PharmaG Ther. 21:371-387, 1983), antiviral protein (13arbieri etal.,Biochem. J. 203:55-59, 1982;Irvinetal.,ArchBiochem. &Biophys. 200:418-425, W 096/33282 -26- PCT~US96/05638 1980; Irvin, Arch Biochem. & Biophys. 169:522-528, 1975), tritin, .shi~pll~ toxin (Caldervvood ef al., PNAS 84:4364-4368, 1987; J~rt ~on et al., Microb. Pafh 2: 147-153, 1987), and Pse~ldQmon~ ~ ~olc,~ A (Carroll and Collier, J. Biol. Chem. 262:8707-8711, 1987).
Wlthin further embo-limPnt~ ofthe invention, ~ ;cpl~e RNA may be utilized as a ~ JAiC gene in order to induce a potent C~ass I ~ e~ on~ Bne~y, m ~d~lifiQn to binding RNA and thereby p~c;vt;l~hilg ~ m of a specific mRNA, high levels of specific ~ e seq~lpnr~es may be utilized to induce the increased cA~resi~;on of i..le.rerons (inr.11l~in~ y-i..le.rt:ron), due to the f~rm~tir~n of large qll~ntitiP~ of double-10 stranded RNA. The increased c,~ ssion of gamma ~~ re~oil (y-IFN), in turn, boosts the c~.lession of MHC Class I ~ntipPn.e Pr~re;~ed ~nti~pn~e seq~lPnc~Ps for use in this regard include actin RNA, myosin RNA, and histone RNA. ~nti~pn~e RNA which forms a ".:~;"~lcl~ with actin RNA is particularly p-c:rt; ~ed.
Wlthin other Pmhot~imPntC ofthe invention, ~ PI~e seq~Pnr,PS are 15 provided which inhibit, for . ~ ~",~ , tumor cell growth, -viral rP~Iir,~ion or a genetic disease by p.~vt;..ling the cellular synthesis of critical pro~eills needed for cell growth.
FY~mpl~PS of such ~ Pn~e sequences include ~ ce thymidine kinase, ~nti~P.n.~e dihydrofolate re~ e ~Maher and Dolnick Arch Biochem. & Biophys. 253:214-220, 1987; Bzik et al., PNAS 84:8360-8364, 1987), ~ p~ee E3ER2 (Coussens et aL, Science 230: 1132-1139, 1985), A~l;c~ e ABL ~F~in~tPin~ et al., Oncogene 4: 1477-1481, 1989), ~nti~Pn~e Myc (Stanton et al., Nature 310:423-425, 1984) and ~nti~Pn~e ras, as well as PI-~e sequ~PncPc which block any ofthe e.~y---es in the nucleotide biosynthetic pdll~wa~
Within other aspects of the invention, gene delivery vehicles are provided which direct the ~ res:iion of a gene product that activates a compound with little or no cytotoxicity (i.e., a "prodrug") into a toxic product. Representative PY~mr1es of such gene products include varicella zoster virus thymidine kinase (VZVI~), herpes .~imrlPY virus thymidine kinase ~HSVI~) (Field etal., J. Gen. Virol. 49:115-124, 1980), andE. coli.
~l~ninpphosrhoribosyl~ sr~ ase(see U.S. Patent Applir~tiQn Serial No. 08/155,944, entitled "Compositions and Methods for Utilizing Con~7itir~n~lly Lethal Genes," filed November 18, 1993; see also WO 93/10218 entitled HVectors Tn~lutlin~ Foreign Genes and W 096/33282 PCTrUS96/05638 Negative ,SPI~cfion MarkersN, WO 93/01281 entitled "Cytosine De~ e Negative SPl~ction System for Gene Transfer Tefhn;ques and Therapiesn, WO 93/08843 entitled "Trapped Cells and Use Thereof as a Drugn, WO 93/08844 entitled "T,~ Cells for the Pluph~laAis or Tre~ -.l of Diseases Caused by ~lruses, Particularly P~thsgf ~ 5 Retrovirusesn, and WO 90/07936 entitled "~eco~h;~ Thel . ~e~ for ~nfPctisn and IIy~G,~roliferative Disol.l~ .") Within prer~ d embof~ ofthe i,l~/cllLoll, the gene delivery vehicle directs the eA~res:iion of a gene product that activates a comro~mf1 with little or no cytotnYif ity into a toxic product in the presel~f e of a p~thn~Pnie agent, thereby loc~li7pd therapy to the pathogenic agent (see U.S. Serial No. 08/155,944).
Within one emho~limpnt of the invention, gene delivery vehicles are provided which direct the cA~,iession of a HSVI K gene duwl~s~lea~ll, and under the l,ansclil Lional control of an H~V plùl"oLer (which is known to be l,~u sclil Lionally silent except when activated by HIV tat protein). Briefly, CAIJleSs..;)n ofthe tat gene product in human cells jnfer~tp~d with HIV and C~ly~lig the gene delivery vehicle causes increased pro~uctirm of HSVI~. The cells (either in vitro or in vivo) are then e -pose~l to a drug such as ~nriclovir~ acyclovir or its ~n~lo~lss ~E;IAU, FIAC, DHPG). Such drugs are known to be pho~h.~ yl~led by HSVI~ (but not by cellular thymidine kinase) to their coll~ onding active n~lr~eoti~e triphosphate forms. Acyclovir and FIAU tripht-sph~tes inhibit cellular polymerases in general, leading to the specific destruction of cells CAlJle~ g HSVI~ in ~ mice (see Borrelli et al., ProG NatL Acad. Sci. USA 85:7572, 1988). Those cells co~ P the gene delivery vehicle and CA~llt;D~ g HIV tat protein are selectively killed by the pl esence of a specific dose of these drugs.
~lthin further aspects of the present invention, gene delivery vehicles of the present invention may also direct the cAI,le:, iion of one or more seqll~ncçs which encode imml-nogenic portions of disease-~eso~i~ted antigens. As utilized within the context ofthe present invention, ~ntig~ne are de~m~-d to be Ndisease-associated" if they are either associated with rendering a cell (or ol~ ~islll) ~ice~eetl~ or are associated with the disease-state in general but are not lc~ ilc;d or .~eetonti~l for rendering the cell ~ ee~ In adAiti~n ~ntig~ne are con~ ered to be "immllnogenicll if they are c~pabl~ under ~plopl;ate contlitione, of causing an immlme l~onse (either cell-m~ ted or humoral).
Tmmllnogenic "portions" may be of variable size, but are preferably at least 9 amino acids long, and may include the entire ~nti~~n W096/33282 PCTrUS96/05638 A wide variety of Hdisease-~Lsori~ted" ~ntigPnx are ~..~ IP~ within the scope ofthe present invention, inr~ ing for eY~mrle immlmngenic~ non-tllmnrigpnic forms of altered cellular components which are normally ~ sori~ted with tumor cells (see U.S.
Serial No. 08/104,424). Rep. cse.,~live ~ ; of altered cellular co- - -po~ which are 5 normally ~sor;~-ted with tumor cells include ras$ (wherein n*n iS understood to refer to ~nti~;enx which have been altered to be non-tllmnrigPnir)~ p53*, Rb*, altered protein encoded by W~ms' tumor gene, ~Ibiqllitin*~ mucin, protein encod~Pd by the DCC. APC, and MCC genes, as well as ,ece~ol:i or rcce~,lor-like structures such as neu, thyroid hormnnP, lCCC~JlOr~ platelet derived growth factor (nPDGFn) ,cc~or, insuLin icceplor, ep:~Prm~
10 growth factor ("EGF") receptor, and the colony stimlll~tin~ factor ("CSFn) lece~,lor.
HDisease-aeeori~tedn ~.,I;g~.~x should also be understood to include all or portions of various eukaryotic (inrl~ing for PY~mr1r, parasites), prokaryotic (e.g, bacterial) or viral pathogens. Re~lcse..~i~c P~ lrc of viral p~tho~n~l include the hçp~titis B virus ("HBVn) and hep~titi~L C virus (nHCVn; see U.S. Serial No. 08/102/132), human p~ri1Qm~ virus (nHPVn; see WO 92/05248; W O 90/10459; ~ O 133,123), Epstein-Barr virus ("EBV"; see EPO 173,254;JP 1,128,788; and U.S. Patent Nos. 4,939,088 and 5,173,414), fe~ne l~k~ virus ("FeLVn; see U.S. Serial No. 07/948,358; EPO 377,842;
W O 90/08832; W O 93/09238), feline immlmodfAfir;rA-nry virus (nEIVn; U.S. Patent No.
5,037,753; W O 92/15684; W O 90/13573; and JP 4,126,085), HTLV I and ~, and human im mlm~drfir~Pnry virus ("HIV~; ~e U.S. Serial No. 07/965,084).
Within other aspects of the present invention, the gene delivery vehicles described above may also direct the ~ ession of one or more ;------- - ~ le ~/Cc~p~ol y mo'eCll~ As utilized herein, the phrase ";.. ~ ccf-exo.~ mole~ Ps" refers to mnlPclllr-s which can either increase or decrease the recognition, pres~pnt~tinn or a.;Livalio of an imm~mP, rc~L,onse (either cell-me~ ted or humoral). Replcsell~ive eY~mrles of ~ cçeeo~ m~leclllPs include IL-1, IL-2,~L-3,1L_4,IL-5,rL~6,rL_7 ~U.S. Patent No. 4,965,195),IL-8,IL~9,IL,lO,IL_ll,IL_12.IL-13,IL,14, and IL,15. C~Volfetal.., J.
Immu~ 46:3074,1991; Gubler et al., PNAS 88:4143,1991; W O 90/05147;EPO 433,827),IL~13 CVVO 94/04680), GM-CSF, M-CSF-1, G-CSF, CD3 ~K~issanen et al., Immunogenetics 26:258-266, 1987), CD8, ICA M-1(S;------~ne et al., Nafure 331:624-627, 1988), ICAM-2 (Singer, Science 255: 1671, 1992), b2-microglobulin ~arnes etal., PNAS
78:2253-22s7~l98l)~LFA-l(Altmannetal.~Nafure338: 521,1989),LFA-3 CWallneret al., J. Exp. Med. 166(4):923-932, 1987), HLA Class I, HLA Class I[ molccllles B7..al1 et al., J. Immun. 143:2714, 1989), and B7-2. Within a ~;r~ed embo-lim~nt 35 the heterologous gene encodes g-IFN.

W 096/33282PCTrUS96/05638 ~.
Within l~-crcl-ed aspects ofthe present invention, the gene delivery velficlcs described herein may direct the ~A~ es:i;oll of more than one heterologous sequPnr-e. Such m~l1tiple seqllpnr~p-c may be controlled either by a single promstPr, or plerclably, by ~rlitign~l spconrl~ry pr"~ lcl:i (e.g, internal ribosome binding sites or "IRBSH). Wlthin s plcrcllcd embo~ ofthe illvenlion~ a gene delivery vehicle directs the cA~Ies~ion of heterologous seq lçnces which act synergistically. For PY~mrl~", within one embo~iimpnt ell~vc~lor constructs are provided W~C~T d~ect ~e CA~IC~;On of a ~ P, such as ~
12, IL 2, y-IFN, or other m~lecllle which acts to il~clease cell-...e~ lP~ pres~ )n in the THl pal}lw~y, along with an immlm~pnic portion of a disease-~ccoc;~ted ~ntigPn In such 10 embo-limpntc i....~ e prts~ ion and proceCcinF~ ofthe disease ~ccori~ted antigen will be i Iclcased due to the prcsence ofthe immllne ~ccesso,y m~leClllP, Wlthin other a_pects ofthe invention, gene delivery vehicles are provided which direct the ~,AI~Ies~ion of one or more heterslos~llc seqUPnr,Ps which encode "rq ~ "~"1 u genes. As utilized herein, it should be understood that the tenn 15 llre~1~ ~P-mPnt genes" refers to a nucleic acid m-~lccnle which f-nco~Pc a Llclapculic protein that is capable of preventing, j"l,.l,;l",g, sti~bili7in~ or reversing an inherited or noninh---rite~
genetic defect. Re~lcsenlal;ve; . les of such genetic defects include disorders in metabolism, i.. --P re~ tinn, hnrmnn~l re~l; tion~ and cll~ylllaLc or IllClll~
s~ccoC;7-te~ structural fimr.ti~m Replcselllali~re; , les of rlicç~cPc caused by such defects 20 include cystic fibrosis (due to a defect in the cystic fibrosis ~ hl~"~e condu~t~nce re~ll~tor ("CFTCR~), see Dorin et al., Na~ure 326:614, ), P~. k;..cG..'s Disease, ~lPnns;
d~P~min~ce defir;-nr,y (NADA"; Hahma et al., J. Bact. 173:3663-3672, 1991), ,B-globin disorders, hemophilia A & B ~Factor VIII-dfAfir;AAnc;ec see Wood et al., Nafure 312:330, 1984), ~llrhPr rlice~ce~ betPc~ forms of gouty a,lL ilis and Lesch-Nylan disease (due to "~RT" dPfir;---nr~es; see Jo~yetal.,PNAS 80:477-481, 1983)Durhpnnp~smllsc~ r dystrophy and familial Ly~ l-ole: ulemia (LDL Receptor mllt~tirnc; see y~m~motQ et al., Cell 39:27-38, 1984).
As is des.i~ ;l.ed herein, T cell pOpl-l~tiQnc trP~ncduced ex ViYo wvith rcLIovi~l vectors ~ ressillg a variety of diLrerellL proLeins can be re-introduced into a patient in order to treat a variety of dirrelc;llL disorders. For ;~ ce, HlV and other viral infectiQnc of T
cells can be treated by this method can be used in the Ll e~ of viral infçction~ of T
cells, inrlu~ing HIV infectionc In particular with regard to ~V infpction~ a number of di~ferenct theraputic approaches can be used. For eY~mpl~, T cells can be tr~n~uced ex vivo with a high titer pl ~ ~Lion of a I t,LI ovi ~I vector l_A~ r essi ~ a nucleic acid or protein which illL~Irelt,s with HIV rep!ic~tio~ (R~ltimore~ D. Nature 335:395, 1988). Inparticular, retroviral vectors cA~I~ssi 1g mutant HlV nucleic acid sequences, ribozymes, W O 96/33282 PC~rrUS96/05638 ce mnlec~ c and ploLeills which can illLt;lrt:l~ with ~V inf~cti~n and replic~tion can be produced as dec~ ed in WO 91/02805, entitled "p~eco~l~h~ RCL1UVhUSeS
Delivclhlg Vector Constructs to Target Cells", and in WO 92/05266, entitled "P~r1~ins~
Cells", both of which p~lblir~tiQnc are hereby L~coll,ul~Ltd by ~crelunce~
S T cell popll1~tionc obLained from patients with a variety of disorders can be tr~ncduced exv~vo with high titer pr~dlions of Ictl UVili~1 vectors ~A~ ill a protein which is effective for ll~~ .1 ofthe disorder when present in the bloodstream. The lecollllfin~ vector construct can express at least one lhcl~l;u~ic protein sPlçcted from the group con.~ ;.-P of factor vm, factor IX, hemoglobin, phenylalanine L~rJlo~ylase, s~d~nosinp~ de~min~p~ hyp~n~ e-~ nin~phn,l~hn~ o~ylLl~l:~rclase~ al-~Lilly~
~n~ .hl~ e conrl~ct~nre re~ll~tor, and ~llcoc~i el~los ~ e The tr~n~ lr,e~l T cells can then be reintroduced into the patient where they secrete the bPnPfiri~l protein into the blood of the patient or the activity of the protein c~etoxifiçs an agent I c*~on:,iblc for the disease (eg. ~dPnnsin~ in ADA ~lPfic;enry or gl~lcoce eberoside in ~-~llrhPl's syndrome).
This approach can be used, for; . '- in the ~l~nl ~ 1 of a variety of genetic disorders, inrlll.1ed those listed above. For ;~ rP~ T cells can be obl~;l-çd from a h_.llophilia patient and tr~n~luced ex vivo with a IcLruvil~l vector ~ ,ressing factor vm. A l~ er of dilrClt;llL factor vm nucleic acid acid constructs can be used. For PY~mp1 -, lcLIuvi~l vectors c,~res:iillg full-length factor vm or a filnr,tion~l factor vm protein lacking the B
domain can be produced as described in Fy~mrhp 2 herein. In ad~lition~ a variety of dirrere..~ retroviral vectors constructs eAl,les~ g full-length factor VIII proteins can be produced as dP~crrihed in co-pending U.S. applir~ation no. 08/366,851, which is hereby inco-~o-~Led by Icrelt:llce.
As dPcrrihed herein, T cells, non-dividing cells, and other cells tr~ tinrl~lly 25 resistant to tr~ncductinn with retroviral vectors can be succPccfillly tr~ncduced ex vivo with high titer plcp~ions of retroviral vectors. In particular, retroviral vectors ~ lessing a protein converting a prodrug to a toxic molecllle can be used alone or in addition to a the ~peuLic protein. As described above, the re-introductinn of cells tr~ncduced with such vectors are useful in the tre~tm~ont of a variety of disorders. In ad~lition this approach can 30 be used to modlll~te the activity oftr~ncduced T cells or other tr~ncduced cells when they are introduced into a patient, by the introduction of the prodrug in vivo.

CA 022l687l l997-lO-l7 W 096/33282 -31- PCT~US96/05638 The term "mod~ te the activity" as used herein, inc~ es the inhil-ition of a ceUularfimr,tionby prodrug ~ lion. Thismnd~ tinnofactivitycanbe ~ccompli~hP~ for eY~mple, by the killing ofthe tr~n~ ced cells by the acLvaled prodrug.
For; . '-, aUogeneic bone nldllUW ~ are used in ~ of cancers such as 5 IP~k~m;~C In ~d~lition~ T cells from the donor are infused in order to aid e.~ n~ and to increase the anti-tumor ;-.. -~ O~P.~ However, a prupol~ion of p~ti~nts treated with this ~11ogenPic ll~ "~;on can develop graft vs. host disease. Eic vivo tr~n~d~ctinn ofthe T cells with relluvi.~l vectors tr~nc~ ce~l with a r~ovil~l vector Pnrorlin~ a protein capable of acliv~ g a prodrug provides a mP~h~ l to mn~ te the 10 activity ofthe T cells after they are introduced into the patient. In particular, the r~s~llt~nt graft versus host disease can be reduced or P~ ed by ~ alion of the prodrug to the patient. A variety of dirr~.~.ll plc,leills, such as herpes thymidine kinase, which are capable of converting a prodrug to a toxic molP~cl-1P~ can be used. Rep~ esenlalive; , ' es of such gene products include varicella zoster virus thymidine kinase (VZVTK), herpes .~imr'~Y virus thymidine kinase (HSVI~) ~Field et al., ~ Gen. Virol. 49: 115-124, 1980), and E. coli. ~nine phosyhcs~ ;bo~yl ~ r~.~se (see U.S. Patent Applir~tinn Serial No.
08/155,944, entitled "composition~ and MPthcl~s for Utilizing Con~litinn~lly Lethal Genes,"
filed November 18, 1993 and Lco.~.o.~ed herein by lert; e ce, see also WO 93/10218 entitled "Vectors Tnr~ inE Foreign Genes and Negative .SPlection Markers", WO 93/01281 20 entitled "Cytosine Dç~min~e Negative Selection System for Gene Transfer Terhni1ues and The. ~-~s", WO 93/08843 entitled NTrapped Cells and Use Thereof as a Drug", WO
93/08844 entitled "Tl~ru---~ll Cells for the Prophylaxis or Tl~ of Diseases Caused by Viruses, Particularly Pathogenic Rellu~uses", and WO 90/07936 entitled~Recollll,...a,ll Therapies for Tnfection and ~Iy~ e Disorders.n) Seq~l~-n~ ~c which encode the above-descrihed heterologous genes may be readily obtained from a variety of sources. For ~Y~mpl~, p1~mids which contain sequ~o-n~ es that encode ;.. ~.e ~cce~o-y molecules may be obtained from a depository such as the American Type Culture Collection (ATCC, Rockville, MD), or from cQmm~rcial sources such as British Bio-Te~hnology T.imited (Cowley, Oxford, Fng]~n~l). Rep-c:se ~lali.~e 30 sources sequ~ncPc which encode the above-noted imm--ne ~ccecco~y mnlecu1es include BBG 12 (co.~ the GM-CSF gene coding for the mature protein of 127 amino acids), BBG 6 (which co~ c sequPnces Pnco~ -~N), ATCC No. 39656 (which CQ..~
sequPnces çnCo~ling TNF), ATCC No. 20663 (which co..l~ c sequ~n~es encot1ing a-lFN), W 096/33282 PCT~US96105638 ATCC Nos. 31902, 31902 and 39517 (which cci. .~ .e sequences Pnro~lin~ b-lFN), ATCC
No 67024 (which co..~ e a sequence which encodes ~1), ATCC Nos. 39405, 39452, 39516, 39626 and 39673 (which co..l~ C seq~lP.ncPe enr~odill~ ~2), ATCC Nos. 59399, 59398, and 67326 (which contain seq~PnrPe çnco~ling IL~3), ATCC No. 57592 (whichS co.. l;~ sequPnc~Ps Pn< orling IL4), ATCC Nos. 59394 and 59395 (which contain seq~lPnres enro-lir~ II~S), and ATCC No. 67153 (which co~ -e seqUPnrPs Pnco~ing IL, 6). It will be evident to one of skill in the art that one may utili~ t;;~ , entir~ se~lu~
ofthe protein, or an ap~lup~i~Le portion thereofwhich Pnrodes the bi~ r~1ly active portion of the protein.
AlLt;~aLively, known cDNA sequ~nres which encode heterologous genes may be obl~i~-ed from cells which express or contain such seqllPncPs Briefly, within one embodiment rnRNA from a cell which c~y.~ses the gene of interest is reverse ~ cc. ;l,ed with reverse ll;1i-ec~ L~ee using oligo dT or random primers. The single stranded cDNA
may then be ~mplifiP,rl by PCR (see U.S. Patent Nos. 4,683,202, 4,683,195 and 4,800,159.
15 See also PCR Terhn~l~ y: Prinripl~e and Applir~tiQne for DNA Amrlifir~tirJn Erlich (ed.), Stor~n Press, 1989 all of which are in.-iol~ul~Led by rerelellce herein in their entirety) utili7ing oli~on~lrleoti~1e primers ccmpl~-nPnt~ry to se~ ces on either side of desired sequ~onrP-e~ In particular, a double stranded DNA is del~Lured by heating in the ~l~;senc.; of heat stable Taq polymerase, seq~ nce specific DNA primers, ATP, CTP, GTP
20 and TTP. Doub~e stranded DNA is produced when ~yl~Lhe~is is ~ ~ . 'cte This cycle may be,~eaLed many tirnes, r~ in a f~CtQri~l ~mplifir~ti~n of the desired DNA.
.Sequ~nr,es which encode the above-desrribed genes may also be sy-nthPei7Prl~ for eY~mplP, on an Applied Bio:iy~L~l"s Inc. DNA ~yl.lhP ;;,~r (e.g., ABI DNA
synthPei7Pr model 392 (Foster City, CA)).
P,e~a,~Lion and Purification of Reco",l,i"a"L Retroviral Particles Another aspect ofthe invention co~ ~.e the pl~h~Lion of l~co...l.;..~ retroviralparticles. P~co...l.i.-~..l reLl~ l particles accor.ling to the invention can be produced in a 30 variety of ways, as those in the art will a~preddLe. For PY~mple, producer cells, i.e., cells col.l;.;..il-E all necpss~ry components for retroviral vector p~ ginE (in~ rlinE a nudeic acid mnipc~ p encoding the retroviral vector), can be grown in roller bottles, in bioreactors, in hollow fiber app~ ~L~Is, and in cell hotels. Cells can be ...~ p~d either on a solid support in liquid mPtlillm or grown as s~ el .e;nne A wide variety of bioreactor35 configurations and sizes can be used in the practice of the present invention.
Cell f~toriPs (also termed "cell hotels") typically contain 2, 10, or 40 trays, are molded from virgin poly:,Lylt;ne, treated to provide a Nuclon D sllrf~cP~, and assembled by sonic welding one to another. Generally, these factories have two port tubes which allow W 096/33282 PCTrUS96/05638 access to the ~ h ~~..be~ ~ for adding reagents or removing culture fluid. A 10-layer factory provides 6000cm2 of surface area for growing cells, roughly the equivalent of 27 T-225 flasks. Cell f~ctori~s are available from a variety of m~n lf~ctl~rers, inr~ ing for ~ pl~
Nunc. Most cell types are capable of pro~-lcin~ high titer vector for 3-6 days, allowing for S mllltiplell~vc;:iLs. Each cell type is tested to dt;~ e the optimal harvest time after seeding and the optimal ~-u---ber of harvest days. Cells are typically initially grown in DMEM suppl~ ~I with 2-20% FB~ in ml}erbott}es until the .~.lu-,cd ~-u--L~r of cells for seeding a cell factory is obl~;..ed. Cells are then seeded into the f~ctc~rips and 2 liters of culture ~upe ~-ala~l~ co~ vector is h~u vt;~ d later at an app~o~.iale time. Fresh media 10 is used to repl~nieh the cultures.
Hollow fiber culture methods may also be used. Briefly, high titer lelluv-~l prodllcti~ using hollow fiber cultures is based on increasing viral cQnr~pntration as the cells are being cultured to a high density in a reduced volume of media. Cells are fed mltriPntc and waste products are diluted using a larger volume of fresh media which circulates 15 through the lumen of ~~u-~eruuS capillary fibers. The cells are cultured on the exterior spaces of the capillary fibers in a bio- ~;lor ~h~".bPr where cell waste products are eYch~nged for mltriPnt~ by rliffi~ n through 30 I~D pores in the capillary fibers.
Retroviruses which are produced from the cell lines are too large to pass through the pores, and thus conce~ le in the hollow fiber bioreactor along side of the cells. The volume of media being cultured on the cell side is a~)p.~x;.. P~ y 10 to 100 fold lower then volumes required for equivalent cell d~n~itips cultured in tissue culture dishes or flasks. This decrease fold in volume inversely correlates with the fold in~uc~i~n of titer when hollow fiber retroviral titers are cG~ ~ed to tissue culture dishes or flasks. This 10-100 fold inrluctiQn in titer is seen when an individual retroviral producer cell line is ~m~n~hle to 25 hollow fiber growth conrlitiQn~ To achieve ..-~ ll l-ll cell density, the individual cells must be able to grow in very close plo~lily and on top of each other. Many cell lines will not grow in this fashion and retroviral p~ ging cell lines based on these types of cell lines may not achieve 10 fold increases in titer. Cell lines which would grow very well would be non-adherent cell line and it is believed that a retroviral producer line based on a non-30 adherent cell line may reach 100 fold increases in titer co...paled to tissue culture dishes andflasks.
Regardless of the retroviral particle and production method, high titer (from about 107-10ll cfu/mL) stocks can be prepared that will cause high level cA~res~ion ofthe desired products upon introductiQn into appropliate cells. When all components required 35 for retroviral particle assembly are present, high-level ~ Iession will occur, thereby producing high titer stocks. And while high titer stocks are plt;rel-ed, retroviral p.t:p~lions having titers ranging from about 103 to 106 cfu/mL may also be employed, W 096/33282 PCTtUS96tO5638 AhhC~llgh retroviral titers can be increased by various purifir;~tinn mPth~c, as described below.
After prodllrti~n by an appro~ means, the infpctis~ls ~t:cc~ cnc IlUpiC
lt;llUVil;Jl particles may be preserved in a crude or pllrified form. Crude re~Ovil~l particles 5 are produced by ~,ulLiv~led infected cells, whelcill re~luvil~l partides are ,el~d from the cells into the culture media. The virus may be p~eSt ~d in crude form by first adding a ~..ll.. - ..1 amount of a fiorml~lAti9n bu ferto the culture ...~lia c~ rthe ,~v~
virus to form an ~qlleollc ~ n R~CO~h;~A~ c~,uvil~l particles can also be plt;sclvc;d in a purified form. More 10 sperificAlly, prior to the Atlrlition of formlllAtisn bu fer, the crude lellùvilal plep~lion desr-rihed above is clarified by passing it through a filter, and then cQnrç~ ed, such as by a cross flow Coll~e~ system ~Filtron Technology Corp., Nol~l~olùugh, MA). ~lthin one embo~imPnt DNase is added to the cOI~c~ e to digest PY~gPnollC DN~ The digest is then diafiltrated to remove excess media conlpol~cll~:j and estAhlieh the ,e~hi~A~l virus 15 in a more desirable bu~elcd so1lltisn The ~1iAfiltr~te is then passed over a gel filtration colllmn such as a Seph ~rlPY S-500 gel cQl~lmn and the purified leCO---~ --A--I virus is eluted.
Crude reconll,il~ tnollupic lclluvill l;,le~ ions can also be purified by ion ~~C1~AI~ge column chlollldLography, such as is d~crrihecl in more detail in U.S.S.N. Serial No. 08/093,436. In general, the crude plcpa.,l~ion is rlr~rified by passing it through a ilter, 20 and the filtrate loaded onto a column CO~llA;-- ~P a highly s~llr~ AI~cd cP~ se matrix, whelch~ the amount of sulfate per gram of cell~ se ranges from about 6- 15 llg. The recc,lllbi l~hlL retrovirus is eluted from the column in purified form by using a high salt buffer. The high salt buffer is then ~ AI~P,ed for a more desirable buffer by passing the eluate over a moleclllAr PYrlllcion col~lmn The purified pl~p~lion may then be 25 formlllAted or stored, preferably at -70~C.
itignAlly~ the pI~ ions CO~IA;I~ g Iecol-~h;~-A~l retroviruses acco~ g to the invention can be conccIlllaLed during pllrific~tisn in order to increase the titer of 1 ~CO' ~ ~1-;l-A ~~1 retrovirus. A wide variety of meth~ls may be utilized for incl easing r eLI ov~l con~P-ntration~ inchl~lin~ for PYAmrl~ pre~ Alisn of Iccc----hil----l Icl-uvi~L~ses with 30 A.~ sulfate, polyethylene glycol ("PEG") col-ce-.l~on, col-cP~ aLion by centrifilg~tisn (either with or without gradients such as PERCOLL, or "cllch;r~nc" such as sucrose, use of con~pntration filters (e.g, Amicon Itration), and 2-phase separations.
Briefly, to accomplish concentration by precipitAtisn of recombinant retroviruses with Amml >nillm sulfate, Ammsnillm sulfate is added slowly to an app, o~,;à~e colu ~ aLion, 35 followed by centrifugation and removal of the Ammsni~lm sulfate either by dialysis or by separation on a hydrophobic colllmn Alternatively, I ecolllbil-a uL I t;ll ~v.~ ~lses may be conc~ aLed from culture,, .P~
with PEG (Green, et al, PN~S 67:385-393, 1970, Syrewicz, et al., Appl. Micro. 24:488-W 096/33282 PCTrUS96/05638 494, 1972). Such mPtho~lc are rapid, simple, and il~,A~ensive. However, like A~
sulfate p~ ip;lAI;on, use of PEG also conc~ les other p-oleil-s from sol!ltinn Wlthin other embo~imPntc, ~ A~ retroviruses may be c~n~ aLed by cPntrifi~g~tion and more particularly, low speed c~ntrifil~tinn which avoids diffirll1tiPC
5 ~c$oci~ted with pplleting that nccc~ A~ c high speed cP.ntrifi~ tinn ~e.g., virus destruction - or inactivation).
Recon~ L l~;L ovil uses accol dmg tcr the invention may a1so be con~ aLed by an P~qlleolls two-phase sepalaLion method. Briefly, polymeric n~leollc two-phase systems may be prepaled by dissolving two di~t; ~llL non-co...l.~l;bl~ polymers in water. Many pairs 10 of water-soluble polymers may be utilized in the construction of such two-phase systems, in~ inE for PY~mp1e polyethylene glycol ("PEGN) or methylc Plllllosç, and dextran or dextran sulfate (see Walter and JO11~I~C5QI~ Anal. Bioc*em. 155:215-242, 1986; A1lJ~;1LSSOI1~
"Partition of Cell Particles and Macromol~c-lles" Wiley, New York 1960). As des-;lil,ed in more detail below in ~yAmple 7, lltili7ing PEG at col-rç..l . aLiOIls ranging from 5% to 8%
(preferably 6.5%), and deYtran sulfate at cQncPntrations ranging from 0.4% to 1%(preferably 0.4%), an ~queolle two-phase system may be e~lAh~ ecl s ble for puliryi~g reco..,l,;..A..I retroviruses. Utilizing such procedures, app~uY;~..AIe 100-fold con~e~ aLon can be achieved with yields of appr.~ .AIf,ly 50% or more ofthe total starting reLIuvi us.
For purposes of illustration, a r~l~senLaLiv-e co--cf~ Lion process which cc~ hi~es several concentration steps is set forth below. Briefiy, l~co.,,l.;.~A~I retroviruses may be plepaled either from roller bottles, cell factories, or bioreactors prior to conc~ aLion.
Removed media CO~IA;~ P the 1eCO~h;I~A~I retrovirus may be frozen at -70~C, or more pl~relably, stored at 2~C to 8~C in large pooled batches prior to proceccin~
For material obtained from a bioreactor, the leco~ retrovirus pool is first ~l~rified through a 0.8 ~m filter (1.2 llm glass fiber pre-filter, 0.8 ~lm ce~ lQse acetate) connected in series with a 0.65 llm filter. This filter &.lA~ .l provides applu~ AIel~ 2 square feet of filter, and allows proceccir~ of about 15-20 liters of pooled material before clogging. For mAt~riAI OtjIA;~d from roller bottles or cell fActQriec a single 0.65 llm cartridge (2 sq. ft.) normally suffices for volumes up to 40 liters. For 80 liter cell factory processes, a 5 sq. ft. filter may be required.
Plc:rtl~bly, after clarifirAtisn the filter is rinsed with buffer (e.g, 150 mM NaCl, 25 mM Tris, pH 7.2-7.5). Following clarifir~tisn, lt;collll~ A~.I retroviruses are cQn~e~ Led by tangential ~ow ultrafiltration utili7ing cAcsettes with a 300,000 mw cut off. For bioreactor material (CO~IIA;";"g 12% to 16% FBS), 4-5 L of material may be c~n~entrated per cAcsette For roller bottles or cell factories at 12-16% FBS, 5-6 L of material may be concentrated per cAccette~ Finally, for cell factories C~I~IA;~;IIg 10% FBS, 8-9 L of mAt~ori may be c~nr~ntrated per cAccette~ Utilizing such procedures at an appropliate pleS:iUlC~
di~rel c;ll~;al between filtrate and 1 el~ .1 Al 1~, Up to 80 liters of material may be cc?l~r~ Led to WO 96/33282 PCT~US96105638 a volume of less th. n 500 mL in under two hours. This process also provides a yield of about 80%.
Following the ultrafiltration step, DNAse may be added to a ~I-c~ aLion of 50 U/mL, and recirculated at a lower pump speed with the filtrate line closed for 30 minllt~e 5 D;eCQ~ OI1e diafiltration is then s~ccomrliehed by adding sd~litiQngl buffer and utili7in~
the same cross di~rere llial ples~u,~ as before. Generally, recovery after this step is applu~ ~ 70%.
Con~e-.l ~ ~led material is then subjected to column ~,l"c..l,~lography on a PhAmsr;s S-500 HG size ~ n gel, ~.I;l;,;.~p, 50 mM NaCl and 25 mM Tris pH 7.2-7.5 as ... ;~;.. salt and ionic ~l-ellgLh conr~ lions. Generally, rtco.. l~ I xenotropic rel.uvi~ses elute offin the first peak.
Tsng~nti~l ilow filtration may once ag. in be utilized to further reduce the volume of the p.ep~lion, after which the conc~ d material is sterilized by filtration through a 0.2 llm Millipore filter.
As an alternative to in vivo prod~lcfion~ the retroviral parl~inP: p.ule"-s may be produced, together or sep~.Lely, from app-~,idle cells. However, instead of introdl~rin~
a nucleic acid mnlecllle enAblin~ prorl~lction ofthe viral vector, an in vifro pa~ ging reaction is conrl~lcted comprising the gag, pol, and env p.u~eins, the retroviral vector, tRNA, and other necPccA~ ~ co""~one"l~. The r~s lhin~ ~t;LIuvh~l particles can then purified 20 and, if desired, conr~ led.

Formulation Of Pharm~ce~tical Compositions Another aspect ofthe invention relates to ph~rm~celltic~1 cv-l,yo~ n~ c~ .y.;~ glt;co."b,n~,L retrovira1 vectors as described above, in co",l)inalion with a ph~rm~ce~tir~11y 5 ~ccept~ble carrier or diluent, while alloll,ef aspect is d-~Led toward a m~thn~l for preserv-ing an i~rrcl;~J~c ~t;c5~ rcl,ovi,uses for s-~bse.~ nt r~cvn~ ;on such that the ,~co...1-i, IA~ rtLlVVllu;~ iS capable of ;- .r~ ., ce}ls upon .~o.. ~
The m~tho~ es~ ~ ;l,ell can be used to preserve a variety of dirr~e~l~ viruses, in~ dinE
.ecc-...hi1~ type C .~I,v~ ses such as gibbon ape le~k~mi~ virus, feline le~ Pmi~ virus 10 and xeno-, poly- and ~. ..phol~ opic murine lellkPmi~ virus (Weiss, et al., RNA Tumor Vimses. 2d ed. 1985). See U.S.S.N. 08/153,342.
ph~rm~ce~1ti~lly acce~ ble carriers or ~ ent~ are nontolriC to le~ at the ~los~g~ and col-c~ lions employed. Rep,cse"l~ e ~ ,les of carriers or rli11lP!nt~ for injectable solllti~n~ include water, i~oton;c saline s~ tiQn~ preferably burre~ed at a 15 physiological pH (such as pho~yhale-l~urrered saline or Tris-l~urre,t;d saline), ...~.~..:1~1, dextrose, glycerol, and eth~nnl as well as polypeptides or p,.)Lei"s such as human serum l1min ~EISA). A particularly p,t:r~;"ed composition comprices a reCol~ A~ enc,l,opic retrovirus in lO mg/mL ..-~ ol, 1 mg/mL HSA, 20 ~I Tris, pH 7.2, and 150 mM NaCI.
In this case, since the ,wo ;nol,opic retroviral particle rcy,esenls applox;...~ y 1 20 ~lg of m~tPri~l it may be less than 1% of high molec~ r weight material, and less than 1/100,000 ofthe total material (inrl~lAing water). This composition is stable at -70~C for at least six mnnthe Pharm~ceutir~l comro~eitione ofthe present invention may also ~d~lition~1ly include factors which stimlll~te T cell division, and hence, uptake and incorporation of vector 25 constructs accord"~g to the invention.
Particularly prcrcllcd methnrle and cornrositione for plesc,v,ng reco...l,;l~
retroviruses are described in U.S.S.N. 08/135,938, filed October 12, 1993, and U.S. Serial No. 8/153,342, filed November 15, 1993.
The use of reco...hil-~ retroviruses to tr~ned~r,e T cells useful in treating patients 30 requires that the product be able to be kansported and stored for long periods at a desired temperature such that infectivity and viability ofthe reco...hi~ retrovirus is ret~inP~l The ~ifficl~lty of p, cse, ving, cco.. .h;~ retroviruses absent low tem~c,~tu,e storage and transport p,csents problems in Third World co-mtriPs, where adequate refrigeration capabilities are often l~rl~ing The initial stabili7~tion of m~t~ri~le in dry form to the preservation of ~ e, slntig~ne and bacteria has been described (Flosodort, et al., J. Immunol., 29:389, 1935).
However, a limit?~tion in this process inrl~ded partial denaturation of proteins when dried from an aqueous state at ambient tempc,~Ltllles. Drying from the frozen state helped reduce W 096/33282 PCT~US96/05638 this denalu~Lion and led to effir;~ nt p, cse v~.~ion of other bi~ pcSIl m~tPri~lc inr~
bacteria and viruses (Stamp, etal., J. Gen. Microbiol., 1:251, 1947; Rowe, etal., Virology, 42:136, 1970; and Rowe, et al., Cryobiology, 8:153, 1971). More recc~lly~ sugars such as sucrose, r~ffin- sP, glucose and trehalose were added in various ~...l.~ ignc as, t~hili7ir~
5 agents prior to Iyophil;~ n of viruses. The use of sugars Pnh~nced lecuvc,~ of viable viruses, for research purposes which require that only some virus survive for later plo~5~l;nn P~coml.;..~..l ,e~luviluses acco,d~lg to the invention can be stored in liquid, or p,cre,~bly, lyophili7P,~l form. Factors inflllpnrin~ stability include the formlll~tion (liquid, 10 freeze dried, con~ Pntc thereof, etc.) and storage cont1itir~nc inrl~ltling ~c~pc~ule, storage co..l;~ , exposure to light, efc. Alternatively, lclluvil~ll particles acco~ g to the invention can be stored as liquids at low le"lpc~ res. In a p, cre" cd embo~limp-nt~ the lcco~ retroviruses ofthe invention are form~ tP~d to plese,vc infectivity in a Iyophili7ed form at elevated ten,pe-~lulcs, and for this form to be suitable for injection into 15 p~tiPntc following Icc0ll~ ;9n ~ co~-.h;.~..l retroviral particles co...~ c~luvi~l vector constructs accor~ g to the invention can be fc - .~ ed in crude or, preferably, pllrifiPcl form. Cmde lC~
~ c~lions may be produced by various cell culture mPth~ s where ~cho~ particles are released from the cells into the culture media. Recc",Li.,~u,l lCL~UVi~;~ll particles may be pl ese. vcd in crude form by adding a sllffi~;~nt amount of r~., .. i~ ion buffer. Typically, the formlll~tion buffer is an ~q~leo~s sQllltion co..l~ g various co...pollenls, such as one or more s~crh~ritles, high m~leclll~r weight structural additives, b~fflering components, and/or amino acids.
The ~ccc,...l~ retroviruses described herein can also be plcsclvcd in a purifiedform. For inet~nce~ prior to the ~ ition of formlll~tion buffer, crude plcp~Llions as described above may be ~ rif e,d by filtration, and then co~c~ ed, such as by a cross flow col- ~ ling system ~Filtron Technology Corp., No. ll,orc,.lgh, MA). DNase may be added to the conc~ le to digest exogenous DNA, followed by diafiltration to remove excess media colllpollents and ~ bsl;l~.~e in a more desirable buffered sollltion The diafiltrate may then passed over a gel filtration colllmn such as a Seph~A~Y~M S-500 gel col--mn, and the eluted ~enol.u~ic retroviral particles retained. A s lffi~;~nt amount of formlll~tion buffer may then be added to the eluate to reach a desired final concentration of the col.J;I~ntc and to Ininim~lly dilute the retroviral p-~alion. The ~q~leoll~ sll~pPn~;on can then be stored, preferably at -70~C, or immf~Ai~t~ly formll1~t~A
In an ~ltern~tive procedure, the crude p-ep~lion can be purified by ion ~ npe column cl~o..lalography. Briefly, the crude reco"lbin~.l retrovirus is clarified by filtration and then loaded onto a column co...~ a highly slllfon~ted c~lllllose matrix. Highly purified reco...billalll xenotropic retrovirus is eluted from the column using a high salt W 096/33282 PCTrUS96/05638 buffer, which is then ~ - ~ h ~ ecl for a more desirable buffer by passing the eluate over a molx~ r ~ oll cQlllmn After r~cu~ely, formlll~tion buffer may the4 added to adjust the final concentration, as rliecllese~l above, followed by low ten~ lule storage, pr~re.~ly at -70~C, or immprli~te formlll~tion S Whenadriedfiormlll~tinnisdesired, anaqueouspl~al~lionco~ p acrudeor purified rell. ~ i plcp&~lioll can be pr~aled by lyophili7~tion or e~,~pol~lioll.
Lyophili7~tion involves cooling the ~leolle ~&alion below thegl~s tr?nei~i~n telnpel~lul e or below the ellte~tic point telll~ re ofthe sol~ltion~ and lelllo~ g water by s~lblim~tion For; , '-, a mllltietep freeze drying procedure as dPs .il,ed by Phillips 10 ef al. (C~yo~iology, vol. 18:414, 1981) can be used to Iyophilize the r,.. l.. l~,~
recol..~in~ll virus, p l~rel~bly from a tempel~ re of-40~C to -45~C. The reel~ltins~
composition should contain less than 10% water by weight. Once Iyophili~p~i~ such a prep~lion is stable and may be stored at -20~C to 25~C.
In an e~ol~ re mPthod, water is removed by evaporation from the retroviral plepal~lion ~q~eol~s s~lepenC;on at ~mhier~t telllpel~lule. E~.~c,l~Lon can be ~co~
by various terhn;~ e~ in~ ing spray drying (see EP 520,748), where the pl~Loll is delivered into a flow of ~ hP~led gas, usually air, wLe~t;u~on water rapidly ~;va~)OI~lCS
from dro~ let~ of the ~.Jspel .c cn Spray drying a~p~lus are available from a l~u~-~be of m~nllfi~ ~ers (e.g, Drytec, Ltd., Tonbridge, Fr~l~nd h_plant~ Ltd., Huddersfield, F.ngl~n(l). Once deLy.ll~led, the l~:con-bill~.l retroviral pl~lion is stable and may be stored at -20~C to 25~C. The res~ltin~ Ule content ofthe dried or lyophili7-e-d ph.~lion may be d~t~ -ed through use of a Karl-Fischer appa,~lus (EM Science ct~r' VlB v~1nmPt-iC titrator, Cherry Hill, NJ), or through a gravimetric mPthrdOnce dehydrated, the rec~ enollopic retrovirus is stable and may be stored at -20~C to 25~C.
As mPntionP,d previously, aqueous pl~lions co~ c;~g xenotropic Ic;l-~viluses accoldi.lg to the invention used for fi~rmlll~tion are typically comrosed of one or more ~.crhs~ni~1Pc high m91cc ~l~r weight structural additives, b~ffe-in~ cQ~ on~ and water, and may also include one or more amino acids. It has been found that the co" ,l~ l ;on of 30 these colllponelll:i acts to preserve the activity ofthe leco~b;~ retrovirus upon Lee~.g and lyophili7~tiQn, or drying through evaporation. See co-owned U.S.S.N. 08/153,342, filed November 15, 1993. Various saerh~rides may be used alone or in co---bin~lion, inrln~lin~ sucrose, Ill~ ol, gIIICOSP~ trph~lose~ inositol, fructose, m~lto~, and ~l~ctosP;
with lactose being particularly prcrc~ d. The conr,çntration ofthe saccharide can range from 0.1% to 30% by weight, preferably from about 1% to 12% by weight. A particularly prert;llt;d concpntration of lactose is 3%-4% by weight. ~itir~n~lly, s~crh~ e cc,llL;..~tic-ne can also be employed, inrl-ldin~ lactose and m~nnitol or sucrose and .;lol It will also be evident to those skilled in the art that it may be plcrt;-~ble to use W 096t33282 PCTrUS96/05638 certain s~crh~ritles in the aqueo--~ sol~lti~n when the lyophili7pd fi~rmlll~til~n is intPnrlP~ for room tclllpclaLllre storage. Sperifir~lly~ tlic~cch~ri~lpc~ such as lactose or trph~lose~ are plcrc~lcd for such form~ tic nc One or more high mo1eclll~r weight structural additives may be used to aid in S p-CVCll~ g retroviral ag~cgalion during Ll~c~g and provides structural support in the lyophili7erl or dried state. In the context of the present invention, structural additives are con~ red to be of Hhigh molec~ r ~ . if ~ ~ *e~ tl.".l 5~0~} c~ A
plercllcd high mnlec~ r weight structural additive is human serum ~Ihllmin (HSA), although other sllhst~nr,es may also be used, such as hydl~AyeLllyl-cP1l~llr~sP,10 Ly~u~ylllethyl-celllllose, dextran, cPll~losP, gelatin, povidone, efc. ~lcrclably, the CQnC~ aLiOn of the high mo~clll~r weight structural additive can range from 0.05% to 20%, with 0.1% to 10% by weight being prercl.cd, and a conCPntration of 0.1% by weight HSA being particularly plcrellèd.
Amino acids, if present, tend to further preserve retroviral illrcc~ivily. In ~d~litil~n~
15 amino acids fim tion to further preserve retroviral infectivity during s~lblim~tinn ofthe cooled aqueous S~ l and while in the lyophili7ed state. A plcrelled amino acid is arginine, but other amino acids such as Iysine, o~ p~ serine, glycine, g]~
asparagine, ~ t~m;c acid or aspartic acid can also be used. Plerel~bly, the amino acid concPntration ranges from 0.1% to 10% by weight. A particularly plcrclled ~f~c 20 concenLIalion is 0.1% by weight.
A variety of b~ . ll'~. ;. .p co---~onellLs may be used to . ~ . a I clalivcl~/ con~ PX
depending on the pH range desired, plererably between 7.0 and 7.8. .~lit~ble buffers include phosph~te buffer and citrate buffer. A particularly plerclled form-ll~ti--n pH is 7.4, and a p-crcllcd buffer is trom~ P~.
It may also be preferable to indude in the f~rmlll~tirn a neutral salt to adjust the f~nal iso-ocmotic salt cQncf~ a~iOn. Sllit~l-lr neutral salts include sodium chloride, pot~cci~lm chloride, and m~P~ chlr~ri~; with sodium chlr~ride being prerclled.
A particularly prerelled method of plèsel Vil~g leco~ ..l retroviruses in a lyophili7ed state for ~ubse~luent Iccol.~ n coll"~lise:j. (a) plcph~ g an aqueous 30 ~ccolllbi~ eLI~lvh~l plcp~alion comprising, in addition to the lécollll~ all~ xenotropic èLIuvilus, about (i) 4% byweight of lactose, (ii) 0.1% byweight of human serum albumin, (iii) 0.03% or less by weight of NaCI, (iv) 0.1% by weight of arginine, and a sllffici~nt amount of 1l u. . .~I hi~ 9 to provide a pH of appl o~ 7.4; (b) cooling the pl ep~ alion to a telllpclaL.lre of about -40~C to -45~C to form a frozen plcp~lion; and (c) removing 35 water from the frozen plepala~ion by sublim~til~n to form a lyophili7~d composition having less than 2% water by weight. It is plerelled that the leco~h;~ xenotropic Icll~v~lls be replic~tiQn defective and suitable for ~ icl~ ion into hllm~nc cells upon lccol~-cl;~ ;on CA 022l687l l997-lO-l7 W 096/33282 PCTrUS96/05638 The lyophi1;~ed or del-yd.~lcd retroviruses ofthe subject invention may be CQ~ ed using a variety of s-1bs~ cee but are preferably lecc~n~ "ed using water. In certain ;~ cee dilute salt So1~ltinne which bring the final rO~ -----1 ~1;nn to is~t~ ~-;ty may a1so be used. In ~d~itinn, it may be adVAnt~geo11c to use ~queo11e SQ1~1tinne C~IIIA;.. ~33 S ciol..~olle-.ls known to el~hAnce the activity ofthe ~;con~ ed virus. Such ~,l.po include CytQl inçe such as Il,2, polycations, such as pro~.~ c sulfate, or other~I-pone.-~s which f-nhA~-re the I~ c~-~ y ofthe le~U..~ ed virus.
Lyophi1i~çd or del-y.ll~lcd leco...hi~A~I virus may be rec~ led with any COIIV~ ~-nt volumeofwaterorthe I~CQI~ 1P agentsnotedabovethatallow~ A..I;~1 and 10 preferably total s~1 1bi1i7~ti~n ofthe lyophili~ed or dehy~lcd sample.

lion of Reco",l,;-,a"l Retroviral Particles In al~OIhCI aspect of the present invention, mrthorle are provided for l.eali-lg human 15 pz~tirnte Aflflirted with a varierty of tliee~eee inr~ ing a genetic r1ieç~eP, cancer, an infectiolle tliee~e~, an A~llo;lllllllme disease, and i.~lli..~..~.~lo~y tlier~eç~ a cardiovascular f1ieç~er and a de~ live ~1ie~ee~ Each of these mçthor1e cQmrri~e ~l...;. .:~;l~ ;.~g to a human a lecolllh~ Vil~ll particle plcp~.lion as described above, such that a thel;~ 1l;r~11y effir~r;o11e ~mmmt ofthe desired gene product(s) enroded by the gene of interest carried on the vector construct is produced. As used herein, a ''Ihcl~l~e~ll;r~lly e fective ~mol1nt" of a gene product e A~-c~ed from a vector construct accG~di~y, to the invention is an ~molmt that achieves a desired therapeutic benefit in a patient to an extent greater than that obsel vcd when the patient was not treated with the gene product. For c~ when the gene product is factor vm, a ''ther~pe~tir-~lly effective ~-,-o-~.l refers to the ~mo11nt offactor VIII needed to produce ll.c.~ .l;r~11y b~-nrfir;~1 r1~ttin~ and will thus generally be dele- ---:--~d by each paLc-~L's ~tten~l;ng physician, ~ltho~gh serum levels of about 0.2 ng/rnL (about 0.1% of "normal" levels) or more will be Llcl~re~ll;r,~l1y b~npfir;~
When the gene product is an RNA moleç~lle with intrisic bir~lo~r~l activity, such an ~ntiernee RNA or ribozyme, a "the.;.l~ l;r~11y effective ~ou~ll' is an amount s11ffiri~nt to achieve a clinically relevant change in the patient's condition through reduced ~,~-c:j~ on of the h~rmfi1l gene product, most often a protein. In a p-cre--ed embotlimrnt~ the RNA
molec111e with intrinsic biological activity will be c,.~.c~:,ed in tr~ned~ced T cells in molar excess to the 1~ ~cled RNA mnlec111e Expression levels of the heterologous and targeted RNAs can be determined by various assays, e.g, by PCR analysis.

3 5 Typical dos~g~s for ex vivo tre~tmrnt of T cells will generally range from about 105 to 1Ol2 infectious 1cco...1~ u.l retroviral particles, with do~e~g~s of 107 to 1010 infectious particles being p-cre~cd. The exact dosage will depend on the ~ul~-l)er of T cells needed for the particular clinical in-lir,~tion and whether the further eyr~n~;~n of the W 096/33282 PCT~US96/05638 -42-tr~ncd~lced and sPlected T cells is re~luhed Thus, the exact dosage for a particular cQn~litiQn can readily be de~ d ~ y.
The volume that the high titer p~ hon of rt;huv.,u:,is delivered in is p.erer~bly not greater than 10% ofthe culture mPrlillm volume ofthe cell culture. More S preferably the volume ofthe high titer r~i;hoviusp,c~honis less than 1%, still more prc:re~bly less than 0.1%, and still more preferably less than 0.01% ofthe total cell culture volume. ~itisn~lly~ the l~L.ùv~sisd~ i~a ~ ~a~ rsfi~cofa~ h6L
disturb or are toxic to the tr~ncd~ced cells in culture (eg. in an ~q~eo~s liquid with a co",pos;Lion similar to that of cell culture ~e T Cells and Non-Dividing Cells Accolding to the present invention, T cells and non-dividing (or "non-rep1ic~ting") cells, or other cells which are ltisi~ to nor nal tr~ncch~ctil~n m~othotls~ are tr~ncd~ced with high effir;~nry using rec~l..bil~ uvh~l particles in ex vivo procedures.
15 Such cells are preferably animal cells, particularly human cells. Upon introd~-ctisn into a patient, the desired gene product(s) rncQded by the vector construct carried by the lchovil~l particles achieve a therapeutic benefit. The tr~ncd~lced cells ~d~ ed to a patient are preferably allogeneic cells, with autologous cells being particularly p~re;~ d.

Various terhn~ es may be employed to s~al~Le the cells by initially removing cells of de~lir,~ted lineage (''lineage-cû.~ ~d" cells). ~nnsr1~-n~l antibodies and mQnor~lc n~l antibody fr~n~ntc are particularly useful for identifying ~llall~c~
~csori~ted wvith particular cell linf~ s and/or stages of dirr~ , ;nn The antibodies (or antibody fr~ mrntc) may be ~tt~rhed to a solid support to allow for crude separation. The 25 separation terhnillues employed should ~x;~ e the viability ofthe fraction to be collected.

W 096/33282PCT~US9~'C-~8 Examples The following ~ . t-s are ineluded to more fully illustrate the present invention.
~iti~n~lly, these i;A 1~' provide plere~led ~mhorlimrntc ofthe invention and are not 5 meant to limit the scope thereo~ St~dald m~oths~lc for many ofthe pl'OC~lU~,s de.e j~ ;I-ed in the following ~ ~~...p'~ ., or s~lit~hlc al~ aL~ procedures, are provided in widely d m~ml~lc of mr1~c~ r biology, such as, for~ntr'~ "Molrvclll~r Clo~
Second Edition (Sambrook et al., Cold Spring Harbor Labol~loly Press, 1987) and "Current Protocols in ~rlcclll~r Biology" (.Al-eub~l et al., eds. Greene ~ceor;~tee/wiley 10 Intersr;~nc~, NY, 1990).

PREPAEU~ON OF T CELLSFOR TR~U~U~11~N
Human leukocyte cell lines were grown in RPMI media supple .~ d with 20%
fetal calf serum; penn/strep; NEAA and L-glu. Cell were grown until they were at a density of appl~Y;...~ely 5Xl05 cells/ml and diluted to lX105 /ml. Cells were tr~ncduc~ in 2 ml volume co-~ 8 ug/ml polybrene and vector added at the moi's in~ir~teA Four to five days later, cells were p~lleted and w~hed in PBS. For lucifer~e assays, cells were Iysed and ~sayed accol-li,lg to m~mlf~chlrer~s instructions (Tropix Inc., Bedford, MA). Beta-gal vector-tr~nedl~r,ed cells were analyzed using the X-gal ~say ~Nolan et al., l9XX).

A . Tr~nechlction of human leukocytes by hi~h titer l ~ll OV;I ~I vectors Various leukocyte cell lines were tested for fi-nrtinn~l tr~neductiQn (i.e., gene c,~ s~ion) with retroviral vectors of varying IIO~iSIIIS. Among those tested were B-gal vectors from two dirrerell~ ~mphotropic and ~noLI opic producer cell lines of canine (DA;
DX; CFA) and human origin (2X), respectively, [DA/CBB gal(V); CFA/ND7(V);
DX/ND7(V); 2X/CBB gal(V)] as well as G-pseudotyped CBB-gal(V) (G-B gal) generated from human 293 2-3 cells. The ampho and xeno vectors were tested at the same titer, all diluted to 1X108 bfu/ml; moi=10, wLel~as the G-vector was used at 10-fold lower concentration, 107 bfu/ml; moi=l (bfiu=blue cell rullllnlg unit and ~IlOi~ tirli~ity Of infection). The frequency of blue cells in each tr~neduced culture is s~ ed below.

3 5 In vitro tr~ne~luction of leukocyte cell lines with vectors of varying 1l OpiSIllS

CA 022l687l l997-lO-l7 W096/33282 PCT~US96105638 Cellline Ce~Type DAJ~g~ CFAJND7 D~UND7 2~g~ G-~g~
Raji Bu~it~l~ L~ ~ + + ++ + +/-HL-60 ~ ~ _ +/_ +/ +/_ SupTl T~ ,' ++ ++ + ++ ++
K562 U ~-~ ~ 11111 11111 1111 111111 1111 CML
U937 I~6Lu~ +/- + +/- + +/-1~ ~t H9 T~elll~ h~..... ~ +/- ++ + +/- +/-OE M Tl~ h.,~ + ++ + +
Hut78 T~llly.l1~h~.. ~ +/- +/- +/- +/- +/-OE M EVT~hybrid + ++ ++~+ 111 +
Xl74 The cell lines were also tested with DAlluci(V), which is a vector p~ Lion encoding the b~ct~ luciferase gene, for relative gene ~ ession. In this c,~ ;",~S parallel cultures were spiked with MA virus to see if lack of luciferace cA~ ,;,;on was at the level of lc;c~Lor tropism, i.e., would helper virus infect the cells and cause a spread of luci(V) leading to greatly h.~.eased ~A~.e.,.,;on of lu~,ir~-~,e. Cultures were tr~n~duced with luci(V) at an moi=5 and MA helper virus at an moi=l . ~t1t1itinn of helper virus to the cultures did not change the luciferase ~ ,sion profiles, either at the level of bULtc protein 10 ~ t;ssion or increase in cellular tropism.

B. Tr~n~duction of plilll~ly cells using high titer rt;lluvil~l vectors P--m~y murine dendritic cells were tr~n~duced using luci(V). The splenic "d~n~lriti~ cell" fraction con~i~tins~ of dendritic cells and maclophag~s was stim~ ted using 15 GM-CSF and murine splenic B+T-lymphocytes were stimlll~ted using con A. After 24 hours, either B-gal(V) or luci(V) was added at an moi=10. The results are shown below in relative light units.

Cells 13-gal(V) luci(V) B+T 300 310 W 096/33282 PCTrUS~6-'C'63 These resutts dPm~ n~ Lle that the splenic dendritic fraction was tr~nQd--Ged by high titer ~. . ~ ,phr.l. opic retroviral vector.

S

PR~PARATION OF RErRovIRAL VECTOR BACKBONES
The following Py~mple d~c~ Q the productinn ofthree l~lovi-~t vector 10 backbones, dçQi~l~tç(l KT-1, KT-3B, KT-3C. Vector KT-l differs from KT-3B and KT-3C in that the former lacks a SF~l~ect~hl~ marker which in KT-3B is neo~ e~ r~"
whereas KT-3C confers phle~sllly~
The Molon~y murine lellkPm;~ virus (MoMLV) 5' long terminat repeat (LTR) EcoR
I-EcoRI L~ l in~ nggagsequ~nceQ fromtheN2 vector (A~ n etal., J. Vir.
61: 1647, 1987; Egtitas et al., Science 230: 1395, 1985) is ligated into the pl~cmid SK+
(Str~t~p~n~, La Jolta, CA). The res -ltin_ construct is deQipn~ted N2R5. The N2R5 construct is ~--~ led by site-directed in vitro mllts~ npQiQ to change the ATG start codon to ATT plevelllillg gag c ~les~"on. This mllt~ni7~d fragment is 200 base pairs (bp) in length and flanked by Pst I restrictiQn sites. The Pst I-Pst I ~ led fragment is pl~rifi~d from the SK+ pl~em;~l and insertêd into the Pst I site of N2 MoMLV 5' LTR in plasmid pUC31 to replace the non-m~t~t~d 200 bp fr~gm~nt The plasmid pUC31 is derived from pUCl9 (Str~t~g~n~ La Jolla, CA) in which ~d~ition~l restriction sites Xho I, Bgl II, BssH
II and Nco I are inserted b~ _n the EcoR I and Sac I sites of the polylinker. This construct is dçeign~ted pUC3 1/N2R5gM.
A 1.0 kilob~ee (Kb) MoMLV 3' LTR EcoR I-EcoR I fragment from N2 is cloned into pl~emi~l SK+ r~s~ltin_ in a construct dçei~ted N2R3-. A 1.0 Kb Cla I-Hind mfragment is purified from this construct.
The Cla I-Cla I do,.i,n~ll s~lect~ble marker gene fragment from pAFVXM
retroviral vector (Kriegler et al., Cell 38:483, 1984; St. Louis et al., PNAS 85:3150, 1988), comprising a SV40 early promoter driving ~,A~.lession ofthe neollly.;il~ (neo) phospho~ r~ se gene, is cloned into the SK+ plasmid. This construct is d~ei~n~ted SK+
SV2-neo A 1.3 Kb Cla I-BstB I gene fragment is purified from the SK+ SV2-neo pl~emi~l KT-3B or KT-l vectors are constructed by a three part ligation in which the Xho I-Cla I fragment co..~ g the gene of interest and the 1.0 Kb MoMLV 3' LTR Cla I-Hind 35 m fragment are il~selled into the Xho I-Hind m site of pUC31/N2RSgM plasmid. This gives a vector dçeign~e(l as having the KT-l backbone. The 1.3 Kb Cla I-BstB I neo gene fragment from the pAFV~I retroviral vector is then inserted into the Cla I site of this W 096/33282 PCTrUS96105638 plasmid in the sense ori~nt~tion to yield a vector deei~ted ae having the KT-3B
backbone.
An ~ ;ve sPlect~blc marker, phleoll,y-,ill reeiet~nce (Mllle~nt et al., Som. Cell andMol. Gen., 14:243, 1988, available from Cayla, Cedex, FR) is used to make the5 l~;;h~vil~l bae~hone KT-3C as follows. The plasmid pUT507 (Mllle~nt, et al., s~pra) is ~igested with Nde I and the ends blunted with Klenow polyl~ se I. The sample is then fi3rther ~lig-peted with Hpa I, Cla I linkers liga~ cu ~ Rr - .... ~ liAh,~
tlig~etinn with Cla I to remove excess Cla I linkers. The 1.2 Kb Cla I L~ c~lyil~8 the RSV LTR and the phleollly-,in le~ e gene is ico1~ted by agarose gel d~llophoresis 10 followed by pllnfic~tion using Gene Clean (BiolOl, San Diego, CA). This fragment is used in place of the 1.3 Kb Cla I-BstB I neoll,y~ r çc~ u~e fragment to give the backbone KT-3C.

1~ PREPARAT~ON OF RETROV~AL VECTOR CONSTRUCTS ENCODING PROTEINS

The following ~y~mple d~e~ es the pl~&~lion of various retroviral vector constructs en~o~lin~ d;~t;l~ human genes of interest. More sperifi~1ly~ part (A) dçs~i~ ;I,es the prod~ction of a vector construct encoding the marker gene ~ tos;d~ce from E.20 coli, part (13) human interferon (hIFN), part (C) a retroviral vector construct ene human int~r1çlllcin-2 (hIL 2), and part ~D) the prod~ion oftwo retroviral vectorconstructs coding for human factor vm. The first factor vm construct, codes for the B
domain deleted fonn of the protein, while the second construct codes for full length factor vm.
A. P, ~al ~lion of ~-gal vectors pCB,13-gal is pltp~d as des~rihed in Irusin et al. (1994) J. Virol. pND7 is obtained by insel ling the E. coli 13-gal into the pNDS (se below) vector af~er ~Y~ ieion of the 30 Factor vm gene.

B . P, epal ~lion of KT-rhY-IFN.

To obtain the human r-IFN gene, the murine homologue is first doned as follows:
35 A my-IFN cDNA is cloned into the EcoR I site of pUCl813 ess~nti~lly as set forth below.
Briefly, pUC1813 (co..~ E a seqll~nre encoding y-IFN) is ~ p~d as eesçnti~lly described by Kay et al., (Nucleic Acids Research 15:2778, 1987; and Gray et al., PNAS

W 096/33282 PCTrUS96/05638 80:5842, 1983). The m~-IFN cDNA is retrieved by EcoR I ~ P~tirJn of pUC18 13, and the i~Ql~tP~d fragment is cloned into the EcoR I site of phosph~t~e-treated pSP73 (P,~,...eg~;
M~rlieon, WI). This construct is d~Pci n~ted SP m~-~N. The oripnt~tion of the cDNA is verified by a~,plc")-iale restriction enzyme dig~sti~m and DNA seq~nrms~ In the sense 5 o, jr .~ n, the 5' end of the cDNA is a~ riPnt to the Xho I site of the pSP73 polylinlcer and the 3' end r~ c~Pnt to the Cla I site. The Xho I-Cla I La~--e--L cQ~ g the m~-IF'N
~Nh in either sense or ~.I;c~ e o- ;~-.1,.1;on i.. .-,L it;ve~ from SPmy-IF~ const~ct an~
cloned into the Xho I-Cla I site ofthe KT-3 retroviral b~r~hon~ This COl~llu~l iS
~P~ign~ted KT m~-IFN.
1. Plcpa~aLion Of Sequences F.nco-lir~ h~-IFN Utilizing PCR

(a) PHA ~fi~nrlnflon Of Jurkat Cells Jurkat cells (T cell line ATCC No. CRL 8163) are rec -crPn-led at a c~ c~ Lion of 1 x 106 cells/ml in RPMI growth media (Irvine Sr;~-ntifir Santa Ana, CA) with 5% fetal bovine serum ~E;BS) to a final volume of 158.0 ml. PhytohPm( ~I.J~;i-il- ("PHA") (Curtis Mathes Sc;~Pntifir" ~oll~tQn~ TX) is added to the ~Sp~ m to a final cQn~ alioll of 1%.
The suspension is i~Cu led at 37~C in 5% C02 overnight. The cells are harvested on the following day and ~ uoted into three 50.0 ml cPntrifi-~ tubes. The three pellets are cc,l,lbined in 50 ml 1x phnsph~te buffered saline (PBS, 145 mM, pH 7.0) and centrifuged at 1000 rpm for 5 ~ es The :,up~ 17...1 is ~leç~ntPd and the cells are washed with 50.0 ml PBS. The cells are collP~cte~l for RNA i~Ql~ti~n (~ A 7~c~7~1f70n The PHA stim~ ted Jurkat cells are resuspended in 22.0 ml ~l~niAinillm so1llti~n (4 M ~l~ni~lini.lm thio~;yallale, 20 mM sodium ~cet~tP, pH 5.2; 0.1 M dithiothreitol, 0.5%
sarcosyl). This cell~ n;din;~m suspension is then passed through a 20 gauge needle six 30 times in order to disrupt cell l-lcl)-~les. A CsCl sQl~ti-:)n (5.7 M CsCl, 0.1 M EDTA) is then overlaid with 11.0 mL ofthe dis.u~led cell-~l~ni~linillm sQl~ltic~n The sol~tir~n is cPntrifi-ged for 24 hours at 28,000 rpm in a SW28.1 rotor (Reel~m~n, Fullerton, CA) at 20~C. After centrifil~ tion the supc ~-alanl is carefully aspirated and the tubes blotted dry.
The pellet is resuspended in a ~l~nirlinillm-Hcl sQllltiQn (7.4 M ~l~ni~linillm-HCl; 25 mM
Tris-HCl, pH 7.5; 5 mM dithiothreitol) to a final volume of 500.0 IlL This sol~ltion is src;l- cd to a microcentrifuge tube. Twelve and one-half microliters of conce ~l~aled Glacial acetic acid (HAc) and 250 Ill of 100% EtOH are added to the microfuge tube. The solution is mixed and stored for several days at -20~C to p-c~ le RNA.

W 096/33282 PCTrUS96/05638 APcer storage, the sol~ltion is centrifuged for 20 mimltes at 14,000 rpm, 4~C. The pellet is then resll~ ded in 75% EtOH and c~ntrifil~;pd for 10 ....~es in a microfuge at 14,000 rprn, 4~C. The pellet is dried by centrifil~tinn under V~LcuUlll, and rC;~s~ p~ in 300 L ~Pi~ni7~ ~ H20. The concentration and purity of the RNA is ti~ .ed by S m~ optical d~nciti~c at 260 and 280 nm.

(c) Rev~rse Tru,~.,,.~hv,.~e~v,, T----. .e.li~t P.i,~ before use, 5.0 1 (3.4 mg/mL) of pllrifi~l Jurkat RNA is heat treated 10 for 5 .......... ~ at 90~C, and then placed on ice. A sol~ltinn of 10.0 111 of 10x PCR buffer (500 mM KCI; 200 mM Tris-HCI, pH 8.4; 25 mM MgCk; 1 mg/ml bovine serum ~lhlln.;~l (BSA)); 10.0 ~1 of 10 mM dATP, 10.0 111 of 10 mM dGTP, 10.0 111 of 10 mM dCTP, 10.0 111 of 10 mM dTTP, 2.5 ,ul RNasin (40,000 U/ml, Plu~llega; M~ on WI) and 33.0 111 DI
H20, is added to the heat treated Jurkat cell RNA. To this sol~ltion 5.0 1ll (108 nmol/mL) (Seq~ nre ID No. 1), and 5.0 ~11 (200,000 U/ml) MoMLV reverse Ll;.i~e~ e (Reth~
Research Labol~L~slies, EC 3.1.27.5, MD) is mixed in a ~ cl ur~ge tube and ilu .~ l ed at room te llpel~ule for 10 ..~ Following the room temperature i--l "bs~ n, therearition ~ ure is ;~ui~lb~led for 1 hour at 37~C, and then inr,~lb~trd for 5 .~ s at 95~C.
The reverse ll~ ;pl;nr~ reaction ~.~lu.c; is then placed on ice in prep~Lion for PCR
(d) PCR Amplif cation The PCR reaction n~Lul c; co. .l ~ 100.0 111 reverse Ll ~ . ;plion re~ction 356.0 1 DI H20; 40.0 1ll 10x PCR buffer; 1.0 111 (137 nmol/mL) V-OLI #5 (Seq~rnr,e ~ No. 2);
0.5 1ll (320 nmol/mL) V-OLI #6 (~eqll~onre ID No. 3), and 2.5 1ll, 5,000 U/ml, Taq polymerase l~EC 2.7.7.7, Perkin-Elmer Cetus, CA). One hundred microliters ofthismixture is aliquoted into each of 5 tubes.

(.Seq~lPnce ID No. 1) 5' - 3': TAA TAA ATA GAT TTA GAT TTA
This primer is comr'~mPnt~ly to a seq~lence of the m~-IFN cDNA 30 base pairs d~w~l~Lleail~ ofthe stop codon.

V (Sequence ID No. 2) 5'-3':GC CTC GAG ACG ATG AAA TAT ACA AGT TAT ATC TTG
This primer is comrlPmPnt~y to the 5' coding region of the my-IFN gene, b~inningat the ATG start codon. The 5' end of the primer contains a Xho I restriction site.

W 096/33282 PCTrUS96/05638 (Seq~lPnr,e ID No. 3) 5' - 3': GA ATC GAT CCA TTA CTG GGA TGC TCT TCG ACC TGG

This primer is cQmplAmPnt~ry to the 3' coding re~on ofthe my-IF'N gene, ending at S the TAA stop codon. The S' end ofthe primer cr...~ e a Cla I restriction site.Each tube was overlaid with 100.0 1ll mineral oil, and placed into a PCR mArhin (Prir~mr Twin Block System, Ericomp, CA). The PCR ~..,~.. re~ll-Atçs the tt; ~ ,.aLul~
ofthe reaction vessel first at 95~ for 1 minute, next at 67~ for 2 ,..;....IPe and f~ally at 72~
for 2 ..~ s This cycle is l~,pe~l~ 40 times. The last cycle re~ll~tes the te...l.w~Lur~ of the reaction vessel first at 95~ for 1 m-inute~ next at 67~ for 2 .~ es and finally at 72~ for 7 minlltPc The comrlPted PCR ~mr1ifiç~tion re?~ctinne are stored at 4~ for 1 month in pre~ Lion for PCR DNA ieQl~til~n (e) ~ f,A~nOfPCRDNA

The ~queo~lc phase from the PCR ~mrlifir,~tinn rear,tione are ~r~r~ d into a single microfuge tube. Fifty microliters of 3 M sodium acetate and 500.0 1ll of chlo.or~,.... ieo~myl alcohol (24:1) is added to the sol~ltion The sQl~ltinn is vo~ ed and then centrifuged at 14,000 rpm at room temperature for 5 .. ;.. ~PS The upper ~ql~eolle phase is l~ rt;..ed to a fresh microfuge tube and 1.0 mL of 100% EtOH is added. This sQl~ltion is inr~lbAted for 4.5 hours at -20~C and then centrifuged at 14,000 rpm for 20 ...;....~es The ~ e~ is ~1eçA-nte~l and the pellet is rinsed with 500.0 ~11 of 70% EtOH.
The pellet is dried by c~ ;r~s~rl;Qn under a vacuum. The ieol~tPA h~-IFN PCR DNA is resuspended in 10.0 111 DI H20.

2. Construction Of h-IFN Retroviral Vec,tors (a) Creation And ~.~ol~ffiQn Of Blunt-Ended hg-IFNPCR D~A
Fr~ ... b The hy-INF PCR DNA is blunt ended using T4 DNA polymerase. Sperifi 10.0 ~1 of PCR ~mplifi~d DNA; 2.0 111, lOx, T4 DNA polymerase buffer (0.33 M Tris-~cet~te, p H 7.9, 0.66 M poL~s~iul.. acetate, 0.10 M m~nrQ;I-m ~r,et~te, S mluI
dithiothreitol, 1 mg/ml bovine serum ~lhllmin (13SA)); 1.0 1ll, 2.5 mM dNTP (a .. I.XLu.t;
equal molar col~re~ ions of dATP~ dGTP~ dTTP and dCTP); 7~0 111 DI H20;
1.0 ~11, 5000 U/mL, Klenow fragment ~EC 2.7.7.7, New F n~l~n~l Biolabs, MA); and 1.0 3000 U/ml, T4 DNA polymerase (E~C 2.7.7.7, New F n~l~n~ Biolabs, MA) are nnLKed W096/33282 PCT~US96/05638 together and inrub~ted at 37~C for 15 .. ~c The reaction mixture is then ;.. ~ ed at room temperature for 40 ..~ les and followed by an ;.t..~ inn at 68~C for 15 ...;....~Pc The blunt ended hy-INF is ico1~ted by agarose gel ele~hopho~c:~;s~ Spe~ifir~lly~ 2.0 111 of loading dye (0.25% blol-lophellol blue; 0.25% xylene cyanol; and 50% glycerol) is 5 added to reaction mixture and 4.0 Ill is loaded into each of S lanes of a 1% agarose/Tris-borate-~TA (TBE) gel co.~ eth;~ lm bromide. Ele~ihopho-e~is ofthe gel is p~. rV....Pd for 1 hour at 100 volts. The ~esired DNA band ~ Y l~y-~F, app.o~ ly 500 base pairs in length, is vicu~ti7pcl under ultraviolet light.
This band is removed from the gel by ele.;~-ophorèlic h~ srer onto NA 45 paper 10 (,SrhlPifh~r and .~rhllPll Keene, NH. The paper is il~ b~led at 68~C for 40 ...;..~lf ~ in 400.0 1ll of high salt NET buffer (1 M NaCl; 0.1 mM FnTA; and 20 mM Tris, pH 8.0) to elute the DNA. The NA 45 paper is removed from solntion and 400.0 Ill of phenol:chloll~r~ l ico~myl alcohol (25:24:1) is added. The sol~lti~n is voll~,Aed and cPntrifil~d at 14,000 for 5 .;~le5 The upper ~queollc phase is h~re - ed to a fresh tube and 400.0 ~Ll of chlo-of~..... ico~myl alcohol (24: 1) is added. The ~-I ALu-~; is vOI l~Aed and centrLfuged for 5 .. l~c The upper a~ueollc phase is ~ relled, a second time, to a fresh tube and 700.0 ,ul of 100% EtOH is added. The tube is ;..~ at -20~C for 3 days.
Following ;-.-~b~7l;on, the DNA is prer ~ lecl from the tube by cr~ . r~ .g~ n for 20 ...;....les at 14,000 rpm. The :ju~ is dec~ntecl and the pellet is rinsed with 500.0 ~l of 70~/O EtOH. The pellet, co.~ g blunt ended hy-IFN DNA, is dried by centrifilp~tion under vacuum and rPs lcpPn-le~ in 50.0 ~ll of DI H20.
The icol~ted blunt ended h~-IFN DNA is phosphorylated using polym~rleotide kinase. Spe~ifi~ y~ 25.0 ~l of blunt-ended h~-IFN DNA, 3.0 ~l of 10A kinase buffer (0.5 M Tris-HCl, pH 7.6; 0.1 M MgC12; 50 mM rlithiothreitol; 1 mM spermidine; 1 mM
~TA), 3.0 ~l of 10 mM ATP, and 1.0 ~11 of T4 polynucleotide kinase (10,000 U/ml, EC
2.7.1.78, New Fngl~nr1 Riolqb~ MD) is mixed and il~ lrd at 37~C for 1 hour 45 ..-;....IP~ The enzyme is then heat inactivated by incnb~tin~E~ at 68~C for 30 r..;....l~s (~) Ligafion Of hr~FNPCR DNA Into The SK+ Vector An SK+ plasmid is ~lig~sted with Hinc II restriction endon~rl~e and purified by agarose gel electrophoresis as described below. Sperifir~lly~ 5.9 Ill (1.7 mg/mL) SK+
plasmid DNA (Str~t~gen~; San Diego, CA); 4.0 ~ll 10x Universal buffer (str~t~n~ San Diego, CA); 30.1 IlIDIH20, and4.0 IllHincII, 10,000U/mL, aremixedinatubeand inc~lb~ted for 7 hours at 37~C. Following ;.. r~ l;Qn 4.0 Ill of loading dye is added to the reaction mixture and 4.0 ~ll of this solution is added to each of 5 lanes of a 1%
agarose/lBE gel CQllIi~ g eth;rlillm bromide. Electrophoresis ofthe gel is pe.ro....ed for CA 022l687l l997-lO-l7 W 096/33282 PCTrUS96/05638 2 hours at 105 volts. The Hinc II cut SK+ pl~m;rt, 2958 base pairs in length, is vi with ultraviolet light. The ~lip~p~ed SK+ pl~rnid is i~ol~ted by gel electrophoresis.
Depht sphorylation of the Hinc II clea~agc site of the pl~ ~m;rt is p~. rl.. ~ using calf;.~ lkalint~ pht)~hA~;.c~ pet~.ifit~lly~ 50.0 ~I ttip;ested SK+ pla~m;r1, 5.0 ~11 1 M
S Tris, pH 8.0; 2.0 ~11 5 n~I EDTA, pH 8.0; 43.0 ~l H20 and 2.0 Ill, 1,000 U/mL, calf ;"I..~il;"iql ph- s~h~l~c~ (nCIPn) (Bo~h.;..g~ ~annhPim ~ntli~n~pnli~, IN) are mixed in a tube and ;.~ I,..Ied at 37~C for 15 ... ~ P~ Folluw,l~g il-~ "l~ 2.0 111 C~P is added. and the so~ ltinn is in. ~Ib-3l~A at 55~C for 90 .~ es Following this il-.i~l,;.l;t~n, 2.5 Ill 20%
sodium dodecyl sulfate (nSDSn), 1.0 Ill 0.5 M ~TA, pH 8.0, and 0.5 111, 20 mg/n~, p.ol~ase K (EC 3.4.21.14, BoPh~ g~r Ma-l.. h~:.. T~ :a"al~c?ti~ are added, and the sQl ~tit n is int-~lb~ted at 55~C for 2 hours. This sol~ti~n is cooled to room tell.pelaLure, and 110.0 111 phenol:chlolul~ o~myl alcohol (25:24:1) is added. The ~ Ulti iS VUll~A~;d and CPntrifil~d at 14,000 rpm for 5 ~ es The upper aqueous phase is ll~l~rt;ll~d to a fresh tube and 200.0 Ill of 100% EtOH is added. This ~ ure is inc~lb~ted at 70~C for 15 mim-tes The tube is cPntrifi-s~od and the pellet is rinsed with 500.0 ~ll of 70% EtOH. The pellet was then dried by centrifi-~tiQn under a vacuum. The depht~spholylaled SK+
plqcm;d is re~ nrled in 40 Ill DI H20.
The hr-INF PCR DNA is ligated into the SK+ plqcm;~l using T4 DNA ligase.
Sperifir-q-lly, 30.0 Ill blunt ended, phosrhsrylated~ hr-IFN PCR DNA reaction ~lixtu~e, 2.0 20 Ill dephosphorylated SK+ plqcm;d and 1.0 ~ll T4 DNA ligase are co...l.i.-Pd in a tube and inrub~q~ted oven~ight at 16~C. DNA was iCQlqtPd using a nlil""cp procedure. Morecperifirqlly, the bqctPriq-l strain DHSa (Gibco BRL, Ga,~ ,l,ulp MD) is ~ sr~lllled with 15.0 Ill of ligation reaction mixture, plated on Luria-Bertani agar plates (LB plates) co..~ qmp;cillin and S-bromo4-chloro-3-indolyl-~-D-g,qlqrito~;de (X-gal, Gold25 Bioterhnology; St. Louis, MO), and inc~lbqtp~ o~ gll~ at 37~C. DNA is ico1qte~1 from white bqcteriq-l col~nies using the procedure desr-ribed by Sambrook et al. (Molecukrr Cloning, Cold Springs Harbor Press, 1989). The plesellce ofthe hr-IFN gene is detPrmined by restriction endom~cleqce cleavage with Xho L Cla L Ava IL Dra L and Ssp I.
The expected Pn-lomlcle~ce restriction cleavage fragment sizes for plqcm;~ls CQ~ g the 30 h~y-IFN gene are presented in Table 2. The icQl-qted DNA pl~qem;d is decignqted SK hr-IFN
and used in constructing the retroviral vectors.

W096/33282 PCT~US96105638 Table 2 Enzyme Fragment Size (bp) S

Xho I and Cla I 500, 2958 AvaII 222, 1307, 1937 DraI 700, 1149, 1500 Ssp I 750, 1296, 2600 (c) Ligaffon Of hrIFNGene Info R~rvv~r~l Vecfor The illlelrelvn gene is ~e~l~ovt;d from SK hr-IFN vector by tiigestisn wvith Xho I and Cla I r~ctriçtion ~nrlonl-ele~ces The res lltin~ fragment co..~ the hr-IFN
20 gene is ~p~ y 500 bp in length, and is icol~ted in a 1% agaroselTBE gel electrophoresis. The Xho I-Cla I hy-IFN fragment is then ligated into the KT-3 rcl~vil~
backbone. This construct is ~1~oci n~ted KT h~-IFN. The structure and pl~sellce eAl le:i:iion of hr-IFN is del~ ed by h ~ ro- . . .;. .~ DH5a bacterial strain with the KT hr-IFN
construct. Spe~ific~lly~ the bacteria is ll ;.. ~r.. P~ with 15.0 ~ll of ligation reaction 25 ll~lule. Thell;~ r)...~edbaçt~ri~lcellsareplatedonLBplatesco.-~ mpiçillin The plates are ine~b~tecl overnight at 37~C and bact~ri~l col~ ni~c are se1ecteA The DNA is icol~te~l as des~rihed in ~b) above, and rli~sted with Xho L Cla L Dra L Nde L and Ssp I.
The expected ~n-lomlclP~ce restriction deavage fragment sizes for pl~cmirlc co.~ . the h~-IFN gene are pl ~sellled in Table 3.

W O 96/33282 PCTrUS96/05638 Table 3 En~yme Fragment Size (bp) Xho I and Cla I 500, 6500 NdeI 1900, 5100 Dra I 692, 2700, 3600 Ssp I 541, 1700, 4700 Sllbseq~l~nt seqll~nrin~ of KT hr-IFN, the retroviral vector, revealed the presence of a one base pair dP1etir~n within the hy-~;N gene. This d~l~ti~-n is reversed using multi-step PCR procedure.

i. Sequence S~cffQn .~eq~onsçs are obt~ined from IBI Pustell seq~l~nee analysis program ant.
Biotech, Inc., New Haven, CT).

The following hr-~;N prirner seqllenres are used:
(Seq~l~nr-e ID No. 4) 5'-3': G CCT CGA GCT CGA GCG ATG AAA TAT ACA AGT TAT ATC TTG
This primer is the sense sequense co~ y to the start codon ATG region ~nrlir~
seven codons u~Ll ~ all~ of hr-IFN gene, and is de~i n~ted hy-IFN lb.
(Seqllense ID No. 5) 5'-3': GTC ATC TCG TTT CTT TTT GTT GCT ATT
This prirner is the anti-sense seq~lense complim~nt~ry to codons 106 to 120 ofthe hy-IFN
gene, and is de~i n~ted hy-IFN Rep B.
(Sequence ID No. 6) 5'-3':AAT AGC AAC AAA AAG AAA CGA GAT GAC
This prirner is the sense seq~lrnse compl;...~ .y to codons 106 to 120 ofthe hy-IFN gene, and is decign~ted hy-IFN Rep A.

W 096/33282 PCTrUS96/05638 (~equPnre ID No. 7) 5'-3': G CAT CGA TAT CGA TCA TTA CTG GGA TGC TCT TCG ACC TCG

This primer is the anti-sense SeqlUPnce CQ~ *~y to the stop codon ATT region and 5 ~ . IP~ seven codons u~ ofthe hy-IFN gene, and is ~Pei~tPd hy-IIiN 3b.

ii. ~itial PCR

A sol~tion of 1 x 106 KT hy-IFN plasmid msl~l-lP,s in 398.0 111, DI H20; 50 ~
lOx PCR buffer (500 mM KCl and 200 mM Tris-HCI, pH 8.4; 25 mM MgC12; 1.0 mg/ml BSA); 5.0 1ll, 2.5 mM dATP; 5.0 ~ll, 2.5 mM dGTP; 5.0 1ll, 2.5 mM dCTP; 5.0 1ll, 2.5 mM
dTTP; 12.0 111, 18.6 nmol/ml, oliEon~lrlçotide h~-IFN lb; 15.0 Ill, 24.6 nmol/ml, oliEon-lrleotide hr-IFN RepB; and 2.5 ~11, Taq poly...,;-~se is mixed in a microfuge tube and 50 ~11 is aLiquoted into 10 tubes. Similarly, a sol~tir~n of 1 x 1o6 KT hy-IFN p!~- 1 m~eclllP~s in 395.0 ~11, DI H20; 50.0 ~11, 10x PCR buffer (500 mM KCl; 200 mM Tris-HCl, pH 8.4; 25 mM MgCk; 1 mg/ml BSA); 5.0 ~ll, 2.5 mM dATP; 5.0 1ll, 2.5 mM dGTP; 5.0 ~11, 2.5 mM dCTP; 5.0 1ll, 2.5 mM dTTP; 13 ~l, 23.4 nmol/ml, o~ nllrlçoti~1e hy-IFN
RepA; 17.011 l, 18.0 nmol/m1, oligs)mlrleQtide hy-IFN 3b; and 2.5 ~I Taq poly..w.~ is mixed in a microfuge tube and 50.0 111 is ~ lloted into 10 tubes. The 20 tubes are placed in a PCR m~rhinç ~Model 9600, Perkin Elmer Cetus; Los Angeles, CA). The PCR
program re~ll~tes the telllpel~.lule ofthe reaction vessel in the first cycle at 94~C for 2 mimltes The next 35 cycles are re~ ted at 94~C for o.s ...;....I~PC then at 55~C for 0.5 Ps and finally at 72~C for 1 m-inute~ The final cycle is ~t;~ ed at 72~C for 1025 minlltçs This cycling program is dPei~n~ted Program 10.
Following PCR ~mplifir~tion 225.0 ~ll of each reaction tube is mixed with 25.0 Ill loading dye (0.25% blo.llophellol blue, 0.25% xylene cyanol and 50% glycerol, agarose gel loading dye) and loaded into the wells of a 2% agarose gel cc,.~ g eth~ lm bromide.
The gel is electrophoresed at appl~.x;...~lPly 90 volts for 1 hour. Ultraviolet light is used to 30 visualize the DNA band separation. Two bands are ieol~te-l one fragment of 250 bp in size and the other of 150 bp in size by electrophoretic ll~rel onto NA 45 paper. Following plerip;li~l;on~ each ofthe two DNA pellets is resuspel1ded in 20.0 I DI H2O and prepared for further PCR ~mrlifir~til~n W 096/33282 PCTrUS96/05638 -55-iii. Annealing and Second Round PCR

A soh-ti~n of 20.0 ~ll of the 150 bp PCR DNA; 20.0 1ll of the 350 bp PCR
DNA: 161.5 Ill, DI H20; 25.0 111, lOx PCR buffer (500 mM KCI; 200 mM Tris-HCI, pH
8.4; 25 mM MgC12; and 1 mg/ml BSA); 2.5 ~11, 2.5 mM dATP; 2.5 111, 2.5 mM dGTP; 2.5 ~11, 2.5 mM dCTP; 2.5 111, 2.5 mM dTTP; and 1.25 ~I Taq polymerase is mixed in a.~lie~uruge tube and 47.3 111 aliquoted into each of 5 tubes. Each tube is placetiin2PCR
m~rhine (Model 9600, Perkin-Elmer-Cetus, CA). The PCRprogl~"re~ tes the te,,,~c,~luie ofthe reaction vessel for 5 cycles at 94~C for 0.5 ~ e The next cycle is re~-l~ted at 55~C for 1 minute. Following this cycle, 1.2 111 hy-IFN lb and 1.5 ~11 h~-IFN
3b are added to the reaction ~ rc. The tubes are then PGR~mrlifip~l using program 10.
The product is de~ (ed rh~-IFN.

iv. Creafion and Isolation of Blunf-Ended rhg-IFNPCR DNA Fra~ment The PCR~mrlifiPd h~ N DNA is blunt ended using T4 polymerase. Specific~lly, 120.0 lli rhy-IFN PCRssl~lti~nis mixed with 1.25 111, 2.5 mM dATP; 1.25 ~11, 2.5 mM
dGTP; 1.25 111, 2.5 mM dCTP; 1.25 1, 2.5 mM dTTP; 1 1, T4 DNA poly,.,c,~se, and 1.0 Ill 20 Klenow fr~rnPnt This ~l~lUIC is ;~c.,l)~led at room te~ cl~lurc for 10 ~ Jl-,~s Following ;..~ b~l;nn, 13.0 ~11 of agarose gel loading dye is added to the Il~IUlC and this s~ tion is loaded into a 1% agarose gel. The gel is electrophoresed at al~lux;~ lely 90 volts for 1 hour. Ultraviolet light is used to visualize the DNA b~ntiing A 500 bp band is ieol~tecl by electrophoretic transfer onto NA 45 paper as desrrihed above. Following pl~c;~ Qn~ the DNA pellet is resll~sp~n~led in 12.0 1 DI H2O.
The ieol~ted 500 bp fragment is blunt ended using T4 polym~rleotide kinase.
Specific~lly, 1.0 mg ofthis fragment is mixed with 1.5 111 lOx kinase buffer (0.5 mM Tris-HCl, pH 7.6; 0.1 mM MgCk; 50 mM .lithi~ll" t;il-ol; 1 mM spermidine; 1 mM EDTA); 1.5 111, 10 mM ATP; and 1.0 111, T4 polym-rleQtide kinase, and inc~b~tpd at 37~C for 30 ~ Ps v. Ligafion of r*y-IFNPCR DNA Into fhe SK+ Yecfor The rh~-IFN PCR DNA is ligated into the SK+ vector. A sQl ltinn of 2.0 ~1 hy-IFNPCR DNA-kinase reaction mixture; 2.0 ~11 CIP treated SK+ vector, and 1.0 ~11, T4 DNA
ligase is inr~lb~ted at 16~C for 4 hours. DHSa bactPri~ is l~ rulllled as described above.

W O 96/33282 PCTrUS96/OS638 vi. Li~afion of hr-IFNGene Into Refroviral Vecfor T iS~ptinn of h~-IFN gene into retroviral vector is p. . r". 1~ .~ as dpc~ribed above.
The new vector is ~leci~te~ KT h~r-IFN.
C. Pl epal ~Lion of KT-hIL 2.

The method for cloning hIL 2 into KT-3 .cL.~,vu~l vector is ecc~ y i~l.ontirql to the procedure for cloning hg-IFN into KT-3, ~-vith the exception that di~c c -L primers are 10 required for ~mplifir~tion ofthe hIL 2 DNA sequrnrs The following h~2 PCR primer seq~nces are used:

V-OLI #55 (.Seq~lPnr,e ID No. 8) 5'-3': ATA AAT AGA AGG CCT GAT ATG
This primer is compl;.. ~.l;.. y to a sequence ofthe hIL 2 cDNA d~JW-- iLlcall- ofthe stop codon.

V-OLI #1 (Sequ~nr,e ID No. 9) 5'-3': GC CTC GAG ACA ATG TAC AGG ATG CAA CTC CTG TCT
This primer is the sense seq~enre ofthe hIL,2 gene co~.pl;~ to the 5' coding region be~ ~ P at the ATG start codon. The 5' end of the primer co. .l ;~;~ .c a Xho I
restriction site.

V-OLI i~2 (.~eq~l~nre ID No. 10) 5'-3': GA ATC GAT TTA TCA AGT CAG TGT TGA GAT GAT GCT
The primer is the anti-sense sequ~nre of the hIL 2 gene cQ~ ~p~ y to the 3' coding region ending at the TAA stop codon. The 5' end of the primer cc,..l ~ c the Cla I
restriction site.

D. P- ~al ~Lion of Factor vm Vectors.

The following is a description of the construction of several retroviral vectorsencoding factor vm. Due to the size of the full length factor vm gene (7,056 bp), 35 p~rl-~ginf~ consLI i~inLs of retroviral vectors and bec~se s~lection for tr~ncd~ced cells is not a requirement for ex vivo hell.dLopoietic stem cell therapy, a retroviral backbone, e.g, KT-1, lacking a s~1ect~hle marker gene is employed.

W 096/33282 PCTrUS96/05638 A gene PncQ-ling full length factor vm can be obt~ined from a variety of sources.
One such source is the plasmid pCIS-F8 ~EP 0 260 148 A2, p~lk1iehed March 3, 1993), which co..~ C a full length factor vm cDNA whose ~A~ ;on is under the control of a CMV major ;~ P~ e-early (CMV MIE) p~u~oler and Pnh~n~Pr. The factor vm cDNA
S cont~inc appr~y;~ y 80 bp of 5' ul~Ll~n~ d ses~nre from the factor vm gene and a 3' wlL.~ ed region of about 500 bp. In ~1~1itirn between the CMV pro."oler and the factor vm sP~l~nre lies a CMV int~r ~ cisn ~ I The cis Pl~m~nt n~ about 280 bp, co~ ;c~s a splice donor site from the CMV major ;~.. ~l;-~e-early promoter about 140 bp u~:iLlw~l of a splice acceptor from an imm-ln~ElnJbl-lin gene, wvith 10 the intervening region being supplied by an Ig variable region intron.

i. Construction of a Plasmid Encoding Rel~ 1 Vector JW-2.

A pl~cmi~l pJW-2, encodinf~ a rt;Llovil~l vector for cA~Iesslllg full length factor vm 15 is constructed using the KT-l backbone from pKT-l. To f~rilit~te di~ ;o~l cloning of the factor vm cDNA insert into pKT-l, the unique Xho I site is collvel Led to a Not I site by site direcLed mllt~gPnPeic The re~ l pl~cmid vector is then opened with Not I and Cla I. pCIS-F8 is tli~P,sted to comrletirJn with Cla I and Eag L for which there are two sites, to release the fragment enr~o~linE full length factor vm. This fragment is then ligated 20 into the Not I/Cla I rPctr cte~l vector to gd~el~Le a plasmid dec;~1ed pJW-2.

ii. Construction of a Plasmid Encodin~ Retroviral Vector ND-5.

A pl~cm;~ vector Pnro-lin~ a Ll~ l;on of about 80% (applux;~ lely 370 bp) of the 3' untr~nel~ted region ofthe factor vm cDNA, cleciEll~ted pND-5, is constructed in a pKT-l vector as follows: ~s described for pJVV-2, the pKT-l vector employed has its Xho I restriction site replaced by that for Not I. The factor vm insert is genel~Led by r~ stinE
pCIS-F8 with Cla I and Xba I, the latter enyme cutting 5' of the factor vm stop codon.
30 The applox;~ ly 7 kb fragment col~ g all but the 3' coding region ofthe factor vm gene is then purified. pCIS-F8 is also dig~sted with Xba I and Pst I to release a 121 bp fragment co.ll~;..;n~ the gene's tel...;..~l;Qn codon. This fragment is also purified and then ligated in a three way ligation with the larger fragment ~ncorling the rest of the factor vm gene and Cla I/Pst I restricted BLUESCRIPT~) KS+ plasmid (Str~t~g~n~, San Diego, CA) 35 to produce a pl~cmid dçcign~ted pND-2.
The unique Sma I site in pND-2 is then ~~h~n~ed to a Cla I site by ligating Cla I
linkers (New F ~l~n~ Biolabs, Beverly, MA) under dilute con~litionc to the blunt ends W 096/33282 PCTrUS96/05638 created by a Sma I digest. After . C~ ;nn and li~tir~n, pl~em;Ae CQ~ two Cla I sites are iAçnfified and dPcign~ted pND-3.
The factor vm seq~rnr-e in pND-3, bolmAed by Cla I sites and co..l;.;~ the full length gene with a l~ n of much of the 3' u"l~ ed region, is cloned as follows 5 into a plasmid barlrhons derived from a Not I/Cla I digest of p~W-l [a pKT-l dc.;vali~_ by cutting at the Xho I site, bl~mtin~ with Klenow, and inserting a Not I linker ~New F.ngl~n-l Biolabs)], which yields a 5.2 kb Not I/Cla I L ~ pCIS-F8 is .,~,~ witlrE~I 2m~
Eco RV and the rPellltir~ fragment of about 4.2 kb, encoding the 5' portion of the full length factor vm gene, is ieol~teA pND-3 is Ai~eted with Eco RV and Cla I and a 3.1 kb 10 fragment is ieQl~ted The two fr~ mf~nteCo~ p portions ofthe factor vm gene are then ligated into the Not I/Cla I Ai~eted vector b~rl hon~ to produce a plasmid deei~ted pND-5.

iii. Construction of a B Domain-deleted Factor vm Vector The precursor DNA for the B-deleted FVm is obtained from Miles L~oldl~
This cA~.res~ion vector is dçei n~ted p25D and has the exact backbone as pCISF8 above.
The Hpa I site at the 3' of the FVm8 cDNA in p25D is m9Aifi9d to Cla-I by ~ lin~rs~
An Acc I to Cla I fragment is clipped out from the modified p25D pl~em;d ThiS~
20 spans the B-domain d~l~o,tion and inrllldpe the entire 3' two-thirds of the cDNA. An Acc I
to Cla I fragment is ,~,,,-uvcd from the p~W-2 above, and l~laccd ~-vith the mnAifis~l B-domain deleted fragment just d~erribeA This construct is deei~n~ted B-del-l.

As those in the art will applc.;dLe, after construction of pl~cmids rnrotlinp rc~,~)vi-~
25 vectors such as those described above, such pl~emi~e can then be used in the produr,tiQn ofvarious cell lines from which infectious rcco~ retroviruses can be prod~ce~l p~r.~in~ Cell Production A. MLV structural ~ene ~,A~ s~ion vectors To decrease the possibility of replic~tion-co,.,~clc,,l virus being generated by genetic interactions between the MLV proviral vector DNA and the structural genes of the p~rlr~gin~
35 cell line ("PCLn), sep~le e,~,ession vectors, each lacking the viral LTR, were generated to express the gaglpol and env genes independently. To further decrease the possibility of homologous ~eco.,lbi.,~lion with MLV vectors and the resll1t~nt gel~ lion of replir~tinn-compl~-m~nt virus, minim~l seq~ nces other than the protein coding sequences were used. In W 096/33282 PCTrUS96/05638 order to express high levels of the MLV structural pluteil-s in the host cells, strong S~ LO1IaI plUlllUIel:i (CMV early and AdS major late plulllù~el i) were ntili7e~1 An eYq-mrle of the construction of a MoMLV gag/pol ~A~ .;on vector pSCV10 follows:
1. The 0.7 Kb HinCII/~nam fragment en~4~ cc ~ the human .;~lo-.-Pg~ virus (CMV) early h~nc.;. ;~ nql pro",ù~er ~Boshart, et al., CeU 41:521, 1985) was icc.~
2. A 5.3 Kb PstI(partial)/ScaI fragment from the MoMLV proviral plqcm: 1 MLV-K ~/Iiller, ef al., Mol. Cell Biol. 5:531, 1985) ~ c~ c~ p the entire gag/pol co~ing regi~
was icol-qte~
3. A 0.35 Kb DraI fragment from SV40 DNA (residues 2717-2363) e~ P
10 the SV40 late ~ sc;,il,Lonal t~- .--;-- 7l ~n signal wac icOlq-tf~

4. Using linkers and other ~ dald reco~ DNA techn~ ec~ the CMV
promoter-MoMLV gag/pol-SV40 tt, ~ ~ ~;- .i I ;on signal was ligated into the bluescript vector SK+
(str~q~tqg~-np~ San Diego, CA).
An eYqmrle ofthe construction of an MLV xenotropic envelope c ~,es~;ol vector follows.
1. A 2.2 Kb NaeI/NheI fragment co..1 s~ the coding region of the ~tnuLlupic envelope obtqined from clone NZB9- 1 (O'Neill, et al., J. Yirol. 53: 100, 1985) was icolqted 2. Using linkers and other ;,L~d~l lt;co...1~ DNA terhn;1uec~ the CMV early promoter and SV40 late t~ l;rm signal ~lPe~-ribed for the gag/pol e~,es:,ion above 20 (pSCV10) were ligated in the order CMV promoter-xeno env-te,...;-.~l;on signal.

B. Host Cell Selection Host cell lines were screened for their ability to effi~ ;ent1y (high titer) rescue a drug r~eietqnceretroviralvectorAalphaN2(A,...~ --o, etal., J. Vir. 61:1647, 1987;andEglitas, et al., Science 230: 139S, 1985) using rep1i~qtion competPnt ~t;Ll o~ us to produce the gag/pol and env structural genes (nMA" virus). Titer was measured from conflllPnt monolayers 16 h aflLer a ~--e~;----~ change by adding filtered Sllp~ al~l~s (0.45 um filters) to 5x104 NIH 3T3 TK-30 cells on a 6 cm tissue culture plate in the plesence of 4 ug/m~ polybrene followed by sPlocti~ n in G418. Among the non-murine cell lines which ~lPmonetrated the ability to p~ r~e MoMLV-based vector with high titre were the cell lines CF2 (canine), D 17 (canine), 293 (human), and HT 1080 (human). These cell lines are pl ere" ed for production of ~ ging and producer cell lines, although many other cells may be tested and sPlected by such means.
C. Generation of P~ck~in~ Cell Lines (i) P~ Lion of gag/pol interrne~ tPs W 096/33282 PCTrUS96/05638 -60- .

As eY~mpl~~ of the genel ~Lion of gag/pol ~l~Le~ I es for PCL prodllctir~n~ D 17(ATCC No. CCL-183), 293 (ATCC No. 1573), and HT1080 (ATCC No. (~CL 121) ce.lls were CO-~ re~ed with 1 ug of the meth~ A~e rÇ~ict~n~ e vector, p~400 (Gra-h-am and van der Eb, Virology 52:456, 1973), and 10 ug of the MoMLV gaglpol CA~les:.ion vector, pSCV10 S (above) by Gq~ m pho ,~h~ l e co-p, e~ n ~ 17 and HT1080, see Gra-h-am and van der Eb, supra), or liroreel;on (293, see Felgner, etal., Proc. Nafl. Acad. Sci., USA ~4:7413, 1987).
After sPlec~ion for ~ ;r~l~d cells in the ~ nce of *e ~gs ~ i~ 2~ n--,ll--)l- "~*~"
individual drug ~e~;aL~ cell colc~n;es were eYp~n~led and analyzed for MoMLV gag/pol CA~ ~e:i~ion by c,~ c~ r reverse l~..cc. .l~ Ace ~RT) activity (~nor1ifi~ from Gof~, et al., J.
10 Virol. 38:239, 1981) and intr~cPlllJl~r p30gag by Western blot using anti-p30 antibodies (goat antiserum #77S000087 from the National Cancer Tnctih.lte). This method i~ntified individual cell clones of each cell type which ~,Ay~essed 10-SOX higher levels of both plOIeil~S co"")a,ed with that of the p~ p. cell line PA317, as shown in Table 4.

W 096/33282 PCTrUS96105638 PROPERTl~;S OF MoMLV GAG/POL-EXPRESSING CFT~T~s RT p30gag LARNL
GFrT. NAME ACIIVll'Y (CPM) EXPRESSION TITRE
(CFU/ML) 3T3 800 - N.D.
PA317 1350 +/- 1.2x 103 D17 800 - N.D.
D174-15 5000 1 1 1 1 1 1.2X104 D17 9020 2000 1 1 1 6.0X 103 D17 9-9 2200 ++ 1.0 X 103 D17 9-16 6100 1 1 1 1 1 1.5 X 104 D17 8-7 4000 - N.D.
HT1080 900 - ND.
HTSCV21 16400 1 1 1 1 1 8.2 X 103 HTS~25 7900 1 1 1 :~.8 X 103 HTSCV42 11600 ++ 8.0 X 102 HTSCV26 4000 - < 10 293 600 - N.D.
293 2-3 6500 l l l l l 7 x 104 293 5-2 7600 1 1 ~ t I N.D.

The biological activity of these proteins was tested by intro~ ing a r~ l vector, LARNL which ~ resses both the ~ . . .phoL~ upic envelope and a Neo+ marker which confers rçei~t~n~e to the drug G418. In every case, co c~ ion of gag/pol in the cell line and er~v from the vector allowed ~ffi~ nt p~tr~ ng ofthe vector as delt;llll,lled by cell-free Ll~lsrt;l of G418 r~eiet~nce to 3T3 cells (titer). Titer was Ille~uled from conflll~nt monolayers 16 h after a me~ m change by adding filtered S~p~ S (0.45 )lm filters) to 5x104 NlH353 TK+ cells on a 6 cm tissue culture plate in the presence of 4 ug/ml polybrene followed by sPlecfion in - G418. ~ignific~ntly~ the vector titers from the cell lines collc;kLLed with the levels of p30gag (Table 4). Since the level of env should be the same in each clone and is coll"~ ble to the level found in PA317 (data not shown), this in~ic~tes that titre was limited by the lower levels of gag/pol in these cells (in~ lin~ PA317). The titre COIl elaLed more closely with the levels of p30gag than with the levels of RT.

W 096/33282 PCT~US96/05638 (ii) Conversion of gag/pol lines into ~e.-ollul-Gpa~ P cell lines.

As c p!es ofthe g~ lion of ~enc,l,opic PCLs, the gag/pol over c~,esso,:j for D17 (4-15) and HT1080 (SCV21) were co-l,~ re~led by the same techniques ~psrrihed above except that 1 ~lg of either the phleo",yci" rPeict~nr-e vector, pUT507 (for SCV21), or the Ly~ullly~ B reCiet~nre marker, pY3 (for 4-15, see P~lorhlin Pr and Di~el~ nl- Mol. Cell Biol. 4:2929, 1984), an~ 10 llg of the xenotropic ~ u~ A~ II vector, p~t (above) was used. Af~er splp~ctinn for l, ;~ rç~il~~ cells in the pl ~sence of phleol,ly. in or hy~ullly~l, lt;~ e~lively, individual drug resi~ cell c~l~niPQ were P~T~n~lPd and analyzed 10 for intr~cPll~ r c~ession of MLV p30gag and gp75enV proteins by Western blot using specific antisera. Clones were idçntified which eA~,essed relatively high levels of both gaglpol and xeno env.
A ~~u~ber of these xenotropic paç~ ing cell lines were tested for their capacity to par~ P rc;l,OVil;11 vectors by mP~ellring titre after the intro~uction of,c;lruv,-~l vectors. The 15 results are presented in Table 5, below.

VECTOR TlTRE ON XENOTROPIC PCLs KT-l TlTRE (CFU/ML) CELL CLONE ON Hl'1080 CELLS

HT1080 SCV21 1.0 x 105 XFl 1.0 x 105 XF7 1.0x 105 XF12 (HX) 4.5 x 105 X6 9.0 X 104 X10 (DX) 1.3 X 105 X23 8.0 X 104 ~i~h.ost titers are obtained when retroviral vectors are introduced into p~ in~
35 cell lines by infection, as opposed to lli... ;r~C~;Qn (Miller, ef al., Somaf. CellMol. Genet., 12:175, 1986). However, the xenotropic p?~ in~ cell lines dPe~rihed herein are blocked for infection by l~co",l)",~ tnC llop;c retroviral particles since the cells express a xenotropic env protein (i.e., "viral ",~e~re e,lce"). To ove,-o~e the problem of "viral W 096/33282 PCTrUS96/05638 intclrercllce~ whereby cell lines c~lcss.ng a ~elloL,opic envelope protein block later infection by xenotropic MLV vectors able to o~}lt;l wisc infect those cell types, vector .
particles c~ .g other viral envelopes (such as VSV-g protein (Florikiewicz, et al., J.
CellBio. 97:1381,1983;andRoman,etal.,Ei~p. CellRes. 1~5:376,1988)whichbindto 5 cell leceL,tol~ other than the AenoL oL,;c leceplol) may be gt lel~Led in the following manner. 10 llg ofthe p1~cm;~ DNA encoding the rcL. ~ l vector construct to be g~d is co-L~ er~led into a cell }ine whrch ~ ~ei high levels of,~ ,vith 10 llg of DNA from which a VSV-g protein is ~ ,rcssed. The r~s~lh~nt vector, ~--~VSV-g protein, is produced tr~n~ ntly in the co-~ xre~ilcd cells. Two days after 10 L~ xrecl;~n, cell free supe- A~ i are added to prospective ~ellcJLr~L c p~r~1nS~ cell lines (which express gag, pol, and env). Cell free ~ pe~ are then collected from the u:mflllrnt monolayers and titered by PCR Cell clones prod~rin~ the highest titers are s~lected as p~r~ ~ cell lines. This procedure is sometimrs referred to "G-hopping."

VII. Alte~ re Viral Vector P~r~ in~ Techniquçs Several ~d~lifiQn~ tPrnAtive ~:iLc~lls can be used to producç ~"1~ Al~l rt;LIuv--uS
particles carrying a vector construct according to the invention. Some of these ~:,Le.lls take advantage of the fact that the insect virus, baculovirus, and the ....... Ali~n viruses, vaccinia and adenovirus, have been ~ rted to make large ~mr llntC of any given protein for which the col.e~ollding gene has been cloned. For PY~mple, see Smith, etal. (Mol. Cell. BioL 3:12, 1983); Piccini, et al. (Meth En~nnology, 153:545, 1987); and M~n~ollr, et al. (Proc. Natl.
Acad. Sci. USA 82: 1359, 1985). These and similar viral vectors can be used to produce 25 pl o~eins in tissue culture cells by insertion of a~,pl upli~Le genes and, hence, could be ~d~rtecl to make retroviral vector particles.
Adenovirus vectors are derived from nuclear re~lic~tinE viruses and can be defective.
Genes can be inserted into vectors and used to express ploLci ls in .. ~.. .-Aii~n cells either by in vitro construction ~Ballay, et al., EMBO J. 4:3861, 1985) or by recollllfillaLion in cells (Thllm mPl, etal.,.~. Mol. AppL Geneffcs 1:435, 1982).
One plere,l~:d methnd is to construct pl~cmitls using the adenovirus Major Late Promoter (MLP) driving: (1) gag/pol, (2) env, (3) a modified viral vector construct. A
modified viral vector construct is possible because the U3 region of the 5' LTR, which cc,. .~ c the viral vector promoter, can be replaced by other promoter ses~l~ncec (see, for PY~mplç, Hartman, Nucl. Acids Res. 16:9345, 1988). This portion will be replaced after one round of reverse transcriptase by the U3 from the 3' LT~
These pl~cmi~lc can then be used to make adenovirus genomes in vitro (Ballay, et al., supra), which are then tl~l:ire~;ted into 293 cells (a human cell line making adenovirus ElA

, W 096133282 PCTrUS96105638 -64-protein), for which the adenoviral vectors are defective, to yield pure stocks of gag/pol, env and leL..,v~l vector carried s_~al~Lely in defective adenovirus vectors Since the titers of such vectors are typically 107-1011/m~, these stocks can be used to infect tissue culture cells ~im~ u~ at high mllltir1irity The cells will then be plugl~ med to produce rtllOv 5 plol~ls and retroviral vector g~nnmPc at high levels Since the adenovirus vectors are defective, no large ~mmlnt~ of direct cell lysis will occur and lt;llov-l~l vectors can be harvested from the cell s~lpe~
Other viral vectors such as those derived from u--lt;lated It;~ vhi51 vectors (e.g, RSV, MMTV or HIV) can be used in the same manner to generate vectors from p~ cells In10 one embo~limpnt~ these adenoviral vectors are used in cQ~ . I;nn with p~ cells, giving rise to retroviral vector prep~lions from plil~y cells Another alternative for making reco ..l.; ~ enoL.opic re~ ~,vil,ll particles is an in vitro p~ gin~ system For PY~mrle, such a system can be employ the following components:
1. gaglpol and env proteins made in the baculovirus system in a similar 15 manner as dç~rihed in Smith, et al., supra, or in other protein productiQn systems, such as yeast or E. coli);
2 vector constructs made using T7 or SP6 ~sc-il,lion ~ ;..-s or other suitable in vilro RNA-ge..~ ing system (see, for; . 'e, Flamant and Sorge, J. Virol.
62:1827, 1988);
3 tRNA made as in (2) or purified from yeast or .. z.. ~.. ~li~n cells;
4 Iiposomes (p-~r~: ~bly with embedded env protein); and 5 cell extract or purified co---pollents (typically from mouse cells) to provide env proce~sin~ and any or other nece~ y cell-derived ~lnctinn~
~ Within this procedure, the co--lponents of (1), (2), and (3) are mixed The env 25 protein, cell extract and pre-liposome mix (in a s~lit~ble solvent) is then added In a plere;l-ed embo~1impnt the env protein is çmhe(1ded in the liposomes prior to adding the resllhing liposome-embedded env to the mixture of (1), (2), and (3) The mix is treated (e.g, by sonic~tion~ te~llp~ e manip~ tic-n or rotary dialysis) to allow en~rsid~tion of the nascent viral particles with lipid plus embedded env protein in a manner similar to that 30 for liposome en~rsi-l~tion of pharrn~eutir~l~ as described in Gould-Fogerite, et al., Anal.
Biochem. 148:15, 1985) This procedure allows the production of high titers of repli~tion inco.llp~LenL recombinant retroviruses wiLllouL cc~ n with pathogenic retroviruses or replic~tiQn-competent retroviruses W 096/33282 PCTrUS96/OS638 D. Detection of Replication Co",pele"l Retroviruses (RCR) The propensity ofthe p~r~ ~in~ cells dp~srribe~ above to e~ e replir~;~tinn co- - ~pet~ vil ~ls may be stringently tested by a variety of rnPthn~1c~ two of which are 5 dçscribed below.

i. T~re Extended S+_~ Assa~r The rytpnrled S+L- assay de~ l--;--F5 whether rep1ir~tir,n cnmpetPnt infectiollc virus 10 is present in the su~ of the cell line of interest. The assay is based on the emriri observation that infectioll~ retroviruses gene-~e foci on the intlir~tor cell line MiCIl (ATCC No. CCL 64.1). The MiCll cell line is derived from the Mv1Lu mink cell line (ATCC No. CCL 64) by tr~nccl~ctirJn with Murine Sarcoma Virus (MSV). It is a non-producer, non-~ ;r~ p~ c;v~ clone co~ p a rer1ir~tirn dere~ilivc; murine 15 sd ~;o---a provirus, S+, but not a replir~tirJn compP~tent murine l~PllkPmi~ pr~vil~ls, L-.
Tnfection of MiCIl cells with replir~ti~m co...p~ retrovirus "activates" the MSV ~PnnmP
to trigger h; l~ .. n~ which results in foci fiorm~tinn Supe~a~ll is r~ /vc;d from the cell line to be tested for ples~lc,; of rP,plir~tinn collll)ele:lll retrovirus and passed through a 0.45 llm filter to remove any cells. On day 1, MvlLu cells are seeded at 1.0 x 105 cells per well (one well per sample to be tested) on a 6 well plate in 2 mL Dulbecco's M~ ifiP~d Eagle Metlillm (DMEM), 10% I;BS and 8 ~lg/mL
polybrene. Mv1Lu cells are plated in the same manner for positive and negative collLI(~ls on separate 6 well plates. The cells are inr~lb~t~pd overnight at 37~C, 10% C02. On day 2, 1.0 mL of test supel--ala~-L is added to the Mv1Lu cells. The negative control plates are inc~lb~ted with 1.0 mL of media. The positive control co~ ; of three t~ ltinn~ (200 focus r". . . ,;.-g units (ffu), 20 ffu and 2 ffu each in 1.0 mL media) of MA virus ~ller, et al., Molec. and Cell Biol., 5:431, 1985) which is added to the cells in the positive control wells. The cells are inr~b~ted overnight. On day 3, the media is aspirated and 3.0 mL of fresh DMEM and 10% FBS is added to the cells. The cells are allowed to grow to confluency and are split 1:10 on day 6 and day 10, amp1ii~ing any replir,~tion competent retrovirus. On day 13, the media on the MvlLu cells is aspirated and 2.0 mL DMEM and 10% FBSiS added to the cells. In ~clrlition, the MiCIl cells are seeded at 1.0 x 105 cells per well in 2.0 mL DMEM, 10% F~3S and 8 llg/mL polybrene. On day 14, the supe---~lanl from the MvlLu cells is ll~.sr~ d to the collt:~.ollding well of the MiCIl cells and inr~lb~tecl overnight at 37~C, 10% CO2. On day 15, the media is aspirated and 3.0 mL of fresh DMEM and 10% ~BS is added to the cells. On day 21, the cells are ry~min~d for focus formation (appe~"-g as clu~leled, refractile cells that O~t;l~OW the monolayer and W 096133282 PCT~US96/05638 -66-remain ~tt~rhed) on the monolayer of cells. The test article is dete",mled to beco.,~ le~l with repli~ti~n Cc~ -.1 retrovirus iffoci appear on the MiCIl cells.

S ii. Co~ul~iv~Lion of Producer Lines and MdH Marker Rescue Assay As an ~ltPrn~te method to test for the p~ ~ af~ ma ~ .v..~
producing cell line, producer cells are cocultivated with an equivalent ,,u,,,ber of Mus dunni cells ~NIH NIAID RethPcd~ MD). Small scale co-cultivations are pe- r~ .ed by mixing of 5.0 x 105 Mus dunni cells with 5.0 x 105 producer cells and seeding the ~ u t; into 10 cm plates (10 mL standard culture media/plate, 4 ~lg/mL polybrene) at day 0. Every 3 1 days the cultures are split at a 1:10 ratio and 5.0 x 105 Mus dunni cells are added to each culture plate to effectively dilute out the producer cell line and provide n~,~;....-... ~mp1ifir~ti~)n of RCR On day 14, culture sllpel~la~ are harvested, passed through a 0.45 ~m cP]1ll1ose-15 acetate filter, and tested in the MdH marker rescue assay. Large scale co-~;ulLiv~lions are p~- rl.. ~--P-d by seeding a II~lUlt; of 1.0 x 108 Mus dunni cells and 1.0 x 108 producer cells into a total oftwenty T-150 flasks (30 mL ,L~ldald culture medialflask 4 llg/rnLpolybrene). Cultures are split at a ratio of 1:10 on days 3, 6, and 13 and at a ratio of 1:20 on day 9. On day 15, the final ~ell~ls are h~ vesled, filtered and a portion of each is 20 tested in the MdH marker rescue assay.
The MdH marker rescue cell line is doned from a pool of Mus dur~2i cells tr~nc~ ced with LHL, a retroviral vector encoding the h~oll,~cin B r~ t~n~e gene(Palmer, ef al., Proc. Naf'l. Aca~ Sci. USA, 84:1055, 1987). The retroviral vector can be rescued from MdH cells upon infpction of the cells with RCR. One mL of test sample is added to a well of a 6-well plate co.. l;.i~ 1 x 105 MdH cells in 2 mL ~ daud culture mPrlillm ~MEM with 10% FBS, 1% 200 mM L-~ P~, 1% non-ÇssPnti~l amino acids) CQ--~ , 4 ~lg/mL polybrene. Media is replaced after 24 hours with standard culture mP~ m without polybrene. Two days later, the entire volume of MdH culture ~u~
is passed through a 0.45 llm cell~lQse-acetate filter and ~ r~llt;d to a well of a 6-well plate co- ~1 ;1il .. . ~~ 5.0 x 104 Mus dunni target cells in 2 mL standard culture mP~ m co..l;~ polybrene. After 24 hours, :iu~e~ l~lls are replaced with standard culture media co.~n ;..i~ 250 ~lg/mL of hyglullly~ ill B and subsequently replaced on days 2 and 5 with media cc,..l;~ -g 200 llg/mL of hy~u~ cill B. Colonies lt;~is~ll to L.y~ulllyc~l B
appear and are vi~ li7ed on day 9 post-se1ection~ by ~ -g with 0.2% Coom~ie blue.

W O 96/33282 PCTrUS96/05638 EXAMPLE S

PRODUCr~ON OF RFS~ M RT~ANT R E ~ OVIRAL P~RTIrT.Fe S The pro~1uction of . cCQ~ 1l 1 ch ovil~l particles cdl ~yih~ vector constructs acco-ding to the invention, reples~ n~;vc ~ . 'çs of which are d~errihed above in r ~ ~ from the human xenotropic and canine h ~ ~ .phol~ ~l ~ t n ;~ cell lines HX
and DA, respectively, is desrr-bed below.

A. Transient Plasmid DNA T.~n:ireclion of Pac~ in~ Cell Lines HX and DA

The p~rl~ng cell line HX or DA is seeded at 5.0 x 105 cells on a 10 cm tissue culture dish on day 1 with DMEM and 10% fetal bovine serum (FBS). On day 2, the media is replaced with 5.0 mL fresh media 4 hours prior to ~nn.~r~ ;on Standard c~lr;~lm phnsrh~te-DNA co-ple~ ne are performed by mixing 40.0 ~112.5 M CaCI2, 10 ~lg of the p1~em;~ enr,o~i~ the vector to be p?,r~gP~.l, and deioni7pd H20 to a total volume of 400 ~11. The DNA-CaC12 sol ltiQne are then added d~u~wlse with cn. .~1 n~l ~it~tinn to 400 ~ul of pl ec~ ;nn buffer (50 mM HEPES-NaOH, pH 7.1; 0.25 M NaCI and 1.5 mM
Na2HP04-NaH2P04). These Illi~lUle:~ are incub~ted at room telllpcldLult; for 10 .. ;.~les The rçslllt~nt fine ple~ es are added to d~clcll~ culture dishes of cells. The cells are in. ~Ibh~ed with the DNA p~c~ n~e overnight at 37~C. On day 3, the media is as~ildlcd and fresh media is added. Supf~ n~ are ~c~llùved on day 4, passed through 0.45 llm filters, and stored at -80~C.

B. Pac~ in~ Cell Line Tr~neduction DA p~r~gin,~ cells are seeded at 1.0 x lû5 cells/3 cm tissue culture dish in 2 mL
DMEM and 10~/O FBS, 4 ,ug/mL polybrene (Sigma, St. Louis, MO) on day 1. On day 2, 3.0 mL, 1.0 mL and 0.2 mL of each of a freshly collected :iu~cllla~ co..~ nil~ VSV-g pseudotyped retroviral particles C~lyill~! the desired vector are added to the HX cells. The cells are inr~1b~ted overnight at 37~C. On day 3, the pools of cells are cloned by limiting dilution by removing the cells from the plate and Cù!~ P the cell suspension, diluting the cells suspensiûn down to 10 cells/mL and adding 0.1 mL to each well (1 cell/well) of a 96 well plate (Corning, Corning, NY). Cells are in~ b~tpd for 14 days at 37~C, 10% C02.
Several clones producing the desired lecollll~in~lL xenotropic retrovirus are s~l~cted and f~Yp~n-led up to 24 well plates, 6 well plates, and finally to 10 cm plates, at which time the W 096133282 PCTrUS96/05638 clones are assayed for eA~ s~ion of the appl ~ ia~e retroviral vector and the s~are collPcte(l and assayed for retroviral titer.
The p~ ging cell line DA may be similarly tr~n~l~ce~ with rt;cQ---hi~ -l rel-uvi al vectors ge~ led by G-hopping.
S Using the procedures above, DA and HX cell lines may be derived that produce leco.~ retroviral vectors with titers greater than or equal to 1 x 106 cfu/mL in culture.

C. Titer Assays Normally vector titers are d~ i.. od by tr~nedu~inn oftarget cells such as HD1080, with ~plûplia~e rlil~ltinnc of a vector pl,~ ion, followed by :lnfihi~?ti~ sPl~inn and counting of:iulvivillg colonies (WO 91/02805). However, l~o~ xenotropic retroviral vectors c~ulyillg a desired vector construct may not include a gene coding for a sPlect~h~ le marker, as may be the case when the vector construct Pn~odçs a large gene of 15 interest, for ;.~ ç~ full length factor vm, titering assays other than those based on selec~ion of drug rcsisL~l~ colon;es are i~uilcd. To this end, antibody and PCR assays, the latter of which is des~ribe(l below, may be employed to d~ ...;..ç leL uv~l vector titer, i.e., the llul~ ei of inf~ctiolls particles co---l..;~ p the ~ lovil~l vectors ofthe illv~ ioll.
While such a PCR assay may be r~.luil ed in the context of a vector lacking a sP1P~''e 20 marker, it is understood that such an assay can be employed for any given vector.
To use PCR to amplify sequences unique to the l~lUV~I vectors of the invention, various primers are required. Such primers can readily be d~oei~Pd by those skilled in the art and will depend on the retroviral vector backbone employed and the compollellL~
thereof, the particular region(s) desired to be ~mplifiçtl etc. Repl~sell~aLiv-ePy~mrle~e of 25 particular primer pairs include those specific for LTR sequences, p~C1~ging signal seq~l~ncPe or other regions ofthe lt;L-uvil~l backbone, and also include primers specific for the gene of interest in the vector. ~ ifion~1 advantages in using such a PCR titering assay include the ability to assay for F~ennmp-l~e~l;~ ,e~ etc.
In the practice of the present invention, the PCR titering assay is pe~ ru....ed by gluwillg a known number of HT1080 cells, typically 1 x 105 cells, tr~n~duced with a retroviral vector capable of directing eA~Ie~ion of the gene of interest on 6-well plates for at least 16 hr. before harvest. The retroviral vectors used for these tr~n~ductions are preferably obtained from cell culture sul)elll~ s. One well per plate is reserved for cell counting. Cells from the other wells are lysed and their CQ~le~ ol~tç~l DNA is prepared using a QIAmp Blood Kit for blood and cell culture PCR (QIAGEN, Inc., ChaL~wulLll, CA). DNAs are resuspended at S x 106 cell equivalents/mL, where one cell equivalent is equal to the DNA content of one cell.

W 096/33282 PCTrUS96/05638 To ç~lc~ te titer, a standard curve is generated using DNA icol~te~ from u~ cd~ced HT1080 cells (negative control) and HT1080 cells tr~n~dllced with a known vector and having one copy of that vec,tor per cell ~,e~-o...r; (positive control), such as may be pl~ed from p~c~ cell lines tr~n~dllce~ with a retroviral vector ~nro~ing a SPlPCl~lblC ms-rkf~r, e.g, n eo.llycill r ~ r~, For both the positive and negative c~--l-uls, DNA is re ~l~enlded at 5 x 106 cell equivalents/mL. The standard curve is gen~ .aled by colllbi~ g di~el~ ~ ofthe positive~* ~ cantrol DNA, while k~;.,g the total amount of DNA CQn-~ l, and amplif~ing specific sequences thel eLoll- by PCR using primers specific to a particular region ofthe rel-uvi~l vector. A leprese ~I~Li~e group of 10 mixtures for genel~ g a standard curve is:

Tube 100% 75% 50% 25% 10% 5% 0% Blank Positive Control (~L) 50 37.5 25 12.5 5 2.5 0 0 Negative Control (~L) 0 12.5 25 37.5 45 47.5 50 0 Distilled water (~lL) 0 0 0 0 0 0 0 50 5.011L from each tube is placed into one of eight reaction tubes (d~plic~tes are also prepa~ed)~ with the rrm~in~er being stored at -20~C. 5.011L from each sample DNApl'èp~lion are placed into their own reaction tubes in d~lplir~te~ PCR reactir~n~ (5011L
20 total volume) are then ~ e~1 by adding 45.011L of a reaction mix co.. l~;.. ;.-~ the following CO..,pOI ents per tube to be tested: 24.5~1L water, 511L lOX reaction PCR
buffer, 4 ~L of 25 n~ MgC12,4 ~L dNTPs (co.~ ;-.g 2.5 nnM of each of dATP, dGTP, dCTP, and dTTP), 511L of primer mix (100 ng or each primer), 0.25~1L TaqStart monorlon~l antibody (Clonterh Labc,.~lo.ies, Inc., Palo Alto, CA), 1.00 IlL TaqStart buffer (Cl( ntech Labs, Inc.), and 0.25~1L AmpliTaq DNA pOly~lel~se ~erkin-Elmer, Inc.,Norwalk CN). Just prior to ~ oti~ the reaction mix to the reaction tubes, 1 IlL of a-32p dCTP (250 ~Ci; 3000 C/mmol, 10 mCi/rnL, Amersham Corp~"~rlin~ton ~eiEht~
is added into the reaction mix. After ~liq lotinE 45.0 IlL the reaction mix into each of the reaction tubes, the tubes are capped and placed into a thermocycler. The particular 30 dena~ ion, ~nnP~linE e1QnE~tion times and temperatures, and number of th~rmncycles will vary depending on size and nucleotide composition of the primer pair used. 20 to 25 amplification thermocycles are then performed. 5 IlL of each reaction is then spotted on DE81 ion eY~h~nE~ chlullla~ography paper (wh~tm~n~ M~i-lston~, FnE]~nrl) and air dried for 10 min. The filter is then washed five times, 100 mL per wash, in 50 mM Na2PO4, pH
35 7, 200 mM NaCI, af~er which it is air dried and then sandwiched in Saran Wrap.
Q~l~ntit~tiQn is pelr~lllled on a PhosphoTm~Eer SI (Molecùlar Dynamics, Sunnyvale, CA).
Filters are typically exposed to a phosphor screen, which stores energy from ioni7in~
radiation, for a suitable period, typically about 120 min. After exposure, the pht sphor W 096/33282 PCTrUS96/05638 screen is SCAnl1Ç~1, whereby light is emitted in proportion to the r~io~ctivity on the original filter. The SÇAI-I~;'Ig results are then downl~aded and plotted on a log scale as cpm (old"~aLe) versus percent positive control DNA (~bscii~). Titers (infectiollc units/mL) for each sample are c AIclllAted by multiplying the l~u~be of cells from which DNA was S icol~tecl by the pcl-~"L~ge (converted to decimal form) d~ ed from the ;,~dald curve based on the ~letested r~lio~ctivity~ divided by the volume of ,.,I,ov"~l vector used to L,;...~ the cells. As will be app,~,~.le~ bythos~e iII the a~ ~hc. ",. Ih.,.~ of d~(e.;l;~
such as colorimetric m.o.thor1c, may be employed to label the ~mp1ifi~d products.

LARGE SCALE PRODUCTION OF ~F~- MRI~ANT XENOTROPIC REIROV~USES

The ~cso.. ~hil~A~ xcncsL~opic lcL o~",uses ofthe invention can be cultivated in a variety of modes, such as in a batch or contim~Q--c mode. In ~d~1hi~n~ various cell culture terhnologies can be employed to produce cQmm~orcial scale q~lAntities ofthe xellc~L~opic lcl-oviluses accord,llg to the invention. Several such terhn~ es are ~ec~. ;I.e~
below, Alth..~lgh others known to those in the art may likewise be employed.
A. ReCG,.,I)i.lalll ReLIUVilUS Production From Hollow Fiber Cultures i. Cul~re Initiafion To initiate a hollow fiber culture, the hollow fiber bioreactor (e.g, ~; Cellco,Inc., ~- ...A~Iuw,-, MD) is first con~liti~n~d for 48 hours prior to seeding by cirnlllAtin~ a run con~litic)n with 100-200 mL of comrl~te growth media at 37~C. The growth media preferably is that to which the cell line has been adapted. All liquids in the ~ when originally shipped should be as~ Lled and replaced with the co p!cte growth media. When 30 seeding the bioreactor, the cells should not have been split more than 48 hours earlier and should be in log growth phase at the time of harvest for the seeding of the ~. The cells typically are harvested by Lly~ A7Al;on and pelleted by cP-ntrifi~g~tiQn. The cell pellet is then rçs l~p~n~ed in 4 mL of 25% pre-con-litioned media and delivered to the extra-capillary space by syringe using the side syringe ports found on the HFB. After seeding the ~, the cells are allowed to adhere for 20 to 30 .. ;.~les before starting the circ~1Atil~n pump. During this time, the media used to con-lition the HFB is replaced with 100-200 mL
of 25% pre-con~itioned media. The circ~lAti~n feed pump is i..;~;A~ed with the starting flow rate set at 25 mL/min. (setting S with 2 long pump pins). After 1 hour from the time of CA 022l687l l997-lO-l7 W 096/33282 PCTrUS96/05638 ,~viL~,hing the pump on, a one rnL sample of media is collected in order to record the initial levels of lactate and a----l.onia On a daily s~hed~ 1 ml ~mpl~s are coll~oct~d every 24 hours to assay for the daily prod~lctiQn of lactate and ~ ~...O~ The initial 100-200 mL of media is ~ ~h~ d with fresh media when lactate levels begin to reach 2 0 g/L (or the 5 equivalent to 22 mM~) The same volume of media is r~l~ until the culture approaches daily levels of 20 mmol/L When daily levels of lactate reach 20 mmol/L, the size of the r~is_. voir bottle is increased to a SO~ ~ bottle c~ P 50~ nL of ~shmedia The fiow feed rate is then increased to 50 rnL/min when the culture begins to produce 2.2 mmol/day of lactate When daily 500 rnL volumes reach 20 mmol/I, of lactate, 10 the original Cellco supplied rescl VUil feeding cap is c ~ d for a larger ~ ,cl voi~ cap ~Unisyn-vender part ~240820) adapted for the Cellco system with the ~dditiQrl of tubing and male luer lock fittings. This rescl vo~ cap will accQ.. od~te 2 liter Corning bottles (To avoid the ~ ~ ~h~l-ge of resc vui- caps during a culture mn, initiate the run with a large reservoir cap which can also ~ )o. L smaller bottle sizes ) When daily lactate readings are 15 assayed and recorded, the daily levels of lactate prod~ctinn ofthe culture can be used to dcLe....,ne when the culture reaches ...~ . cell density, i.e., when the rate of lactate de.;.cases and levels o~

ii Seeding Density for the 2X-,~-gal To ect~klich specific seeding re4u..c...c..Li, two hollow fiber runs are pc-ru~ cd, one run seeded with a low ~-un-bcr of cells, the other seeded with a high ...l...l~er of cells Progress of each culture is tracked by analyzing the daily glucose cc-~ pLion and lactate 25 pro~lction levels.
In this experiment, one HFB was seeded with 1 3 x 107 cells (repres~ p the low seed culture), the other with 1 6 x 108 cells. Here, the cell line 2x-B-GAL17 14 was able to initiate a good hollow fiber run under both seeding conrlitiQnc T~.;l;5.l;,-p, a run with fewer cells is primarily convenient for red-lcing the effort ~c~ ired for gcn~-~L-ng the number of 30 cells required to start a culture, ~ltho--gh fewer cells initially extends the time it takes to reach optimal cell d~-n~iti~, which usually yield the highest titers 2x-B-GAL17 14 adapted well to hollow fiber culture, eventually requiring daily media çh~l~pes of 500 rnL in order to avoid ~ccllm~ tion of toxic levels of lactate Pl~te~l linp~ of daily lactate pro~-ction and drops in peak titer prodoction correlated with ~nX;~ cell cl~n~iti~s and the relative 3 5 health of the culture iii. Opfimal Titer CC r~Ct ,.tr~tions. Frequencv of Harvests and Tofal W 096/33282 PCTrUS96/05638 Harvest Amounfs B-gal titers for the above CA~C~ l were del~ d from frozen ~ on 293 cells assayed 48 or 72 hours after tr~ncd~ on The tr~n~~ cells were stained for ~gal 5 activity- and cu~ ed on a hemo~;ylc,.,clcr to yield a titer based on the ~u~ er of blue cells /mL (BCT/mL). O~l-,--u--- titers were generally oblAi~ d on day 7 of a high seed culture at ~.8 X108 BCT/mL from a 72 hour blue cell titer otr 293 ce~s. A ~h,~i- ~ cc~t~i~rseeded at a 10 fold lower seeding density peaked at 5.2 x107 BCT/mL from a 48 hour blue cell titer. Co-.-pOled to flat stock cultures (from tissue culture dishes or fiasks) titered using 48 hour blue cell titers on Hl 1080 cells (ç~lr, ll~ted to be about 5 X106 BCT/mL), the increase in titer by using hollow fiber :iy~e...s is appl.~x;...P ~ely ten fold higher. These ... ;~ Y ;.. titers observed were reached prior to hitting 20 mmollL lactate levels, which ~pea. ed to reduce titers produced the following week.
Crude ~lp~ ..A~A..~i can be h~vt;~led every 9 hours with out any loss oftiter and 15 three h~ .~ per day should be po~ '~ with ..-;~ -.. titre loss. In ~dditi~n~ co..~
hollow fiber cultures can be ~--~ ed for several weeks. When titers were co---p~ed belw~ll the low and the high seed culture, there was little di~t;-t;nces by day 11 belw~n the two seed cultures, both of which av~ ~ed 4 x 107 BCT/mL.

TWO-PHASEPURUFICA~ON OF ~F~' ~Rn~ANTR~RovlRusEs A. Col~ce~ lion of DA/ND-7 ~cCOIllbil~alll p~Li~,les 1400 ml of media (DME~I co.~ P. 5% Fetal Bovine Serum) co.~ g DX/ND-7 vector at a titer of 1.25 x 106 cfil/m~ iS used as starting m~t~ l Three }~un-l~cd ~ni~ t~rs of two-phase partitit~nin~ CO",pOl-clll:~ (PEG-8000 (autoclaved), dextran-sulfate, and NaCl) are added to a final con~ Lion of 6.5% PEG, 0.4% dextran- ~ h~ l e~ and 0.3 M NaCl.
The resl-lt~nt s~1~ltir)n is placed into a t.wo-liter separatory funnel, and left in a cold room for 24 hours (in~ ling two mixing steps app,ux;.. ~lel~ 6 to 16 hours apart).
Following the 24 hour period, the bottom layer (app~ux;lll~tply 20 mL) is carefully eluted, and the interphase (apprv~ ly 1 mL) is collected in a 15 mL conical FALCON
tube. The interphase co..~ g vector is diluted to 10 mL by addition of PBS, and in~b~ted at 37~C in order to bring the sollltion to room Ic,,,~,c-~Lu-c and d~et~hili~e the 3 5 micellPs To one-half of the diluted interphase, KCl is added to a final con~entration 0.4 M, and mixed well. The tube is then placed on ice for ten .~.;..u~e-~, and spun for 2 mim'tes at 2,000 rpm in a bench-top centrifuge. The su~ ..1 is removed and filtered through a W 096/33282 PCTrUS96105638 0.45 ~m syringe filter. The other half of the "lLel~hase co. .1~ vector is sepa.~Led by S-500 ,SeFh~ Y clll-,lllalography in lX PBS. The results ofthese CQnC~ ion proce~s~s, as determined in a BCFU assay, are shown below in Table 6:

S

PHASE OUANlITY OF VECTOR
Crude 1.75 x 109 bcfu Separation: Top phase 1.4 x 108 bcfu Separation: Interphase 7(+/-3) x 108 bcfu Separation: Bottom phase 2 x 106 bcfu Final step: KCI sepal~lioll *6(+/-3) x 108 bcfu Final step: S-500 sep~lion *1.8(+/-0.3) x 108 bcfu * Note that since the sample was split into two halves, that these ~lullLcl:i were d: ~'e d in order to 1 ~pl t:Selll the level of p~lrific~ti~n that would be ~,A~e-;led if the entire 1 mL
interphase was sep~ualed as in~irslSe~
In ~ y, 1.4 liters of crude It;sear ch grade :iupe~ .ll co~ recollll)ina ll retroviral particles may be reduced to a 10 mL volume, with appruX;~ y 50% (+/-20%) being recovered when KCI sepal alion is utilized as the final step. When S-500 cLlulllalography is utilized as the final step, only about 10% ofthe initial rec~
retroviral particles are recovered in a 14 mL.
In order to comrlrvte cc ~ e-~l~ aLiOll ofthe retroviral vector particles, the vector-CG~ solution may be further subjected to CQ~ alion lltili7.in~ an MY-,ll~,.,~.a.~e Amicon filter, thereby red~l~inp the volume from 10 to 14 mL, down to less than 1 mL.

PRODUCTION OF VECTOR FROM DX~ND7 B-GAL CLONE 87 UTIL~NG A CF.T T FACTORY

DX/ND7 bgal clone 87, an ~,A~,ession vector, was grown in cell f~ctQries Cells 25 were grown in DMEM suppl~m~nted with Fetal Bovine Serum in roller bottles until enough cells to seed 20 10-layer cell factories (NUNC) at a 1:3 dilution were obtained.
Each 10-layer cell factory is seeded with appru~ ly 0.8 liters of cell ...~,1;...~.
Cells were seeded into the cell factory by pouring media co~ cells into the factory so that the s~lsp~oneion~ evenly fill the 10 layers. The factory is then carefully tilted w 096/33282 PCTrUS96/05638 away from the port side to prevent the ~ o~- from re~ trihlltir)n in the co..--.-~ tube.
Finally, the cell factory is rotated into its final upright position A h~vel~L filter is ~tt~-he~l to each port. The factory was then placed in a CO2 ;..~ or.
In three days, and for each of the next three days, S~ CO~ g vector 5 was harvested. The cell factory is pl_ced in a tissue culture hood. One filter is r~;n.oved and sterile 1.~. ,re, tubing is cnl~ td to the open port. The factory is lifted so that :jupe~,.ala~-l d~ains into the tubing. App.,~ sof i,~.~,. . . A$A. A is ha~ ._ .~1 fi~
each factory. Fresh DMEM/EBS is used to repl~n;~h the lost ~ i;.. The l.~re, tubing is removed and the factory r~laced in the i~ b~or. From 20 cell fi-A~ori~c applu~
10 90 liters of crude vector co~ ;--g ~u~e- ~ were ob~ A
V~rifi~tion ofthe vector was performed by trAn~ ction of HT1080 cells. These cells were h~ ~ ed 2 days later and stained for b-gal protein. The titer of the ~ I A. .I was d~ .ecl to be 2 x 107/ml.

CONCENTRAIION OF~F~Rn~ANTR~E~ROVIRUSBY LOW-SPEEDCEP~R~FJGA~ON
20 A. Retrovector SupelllaL~Il Pl~p~ ion Producer cell lines DA/Bgal and ~DN-7 were cultured in a culture flask and a roller bottle, .~I.e.Livel~, CO~ Dulbecco's Modified Eagle's MP~ m (DMBM) supplPmPnted with 10% fetal bovine serum plus lmM ~t~ t~Aminp~ Sodium pyruvate, non-25 eC~pntiAl amino acids and antibiotics. Viral :iu~ L~ was harvested from the flask androller bottle, and were filtered through a 0.45 um syringe filter. The filtered S11P~
were stored either at 4~C (EWND7), or frozen at -70~C ~DAB-gal).

B. Virus ConcenL"lLion Viral SU~t;111aLaI1l was aliquoted into 50 ml sterile OAKRIDGE screw cap tubes, and placed into an SS34 rotor for use in a Sorvall cPntrifilge The tubes were spun for 1 hour at 16,000 rpm (25,000g-force) at 4~C. Upon completion ofthe spin, the tubes were removed, the supeIllal~ll deçAnted and a small opaque pellet res~lspend~Pd in the DMEM
35 media desçrihed above.

C. Virus Titration W 096133282 PCTrUS96/05638 Co~ Led virus was titered on HT1080 cells plated 24 hours earlier at a cell density of 2xlO5 cells per well in a six well plate + 4 llg/ml polybrene. Briefly, virus preps were diluted from 1/10 to 1/10,000 and 50 111 of each ~ utic~n was used to infect one well from the six well plate. Plates were ;~,ub~ overnight at 37~C. Forty-eig~ht hours later, S cells were fixed and stained with X-gal. The results are set forth below in Table 7.
-Table 7. Virus COI~Cfn~ .Lon LLuugll Low Speed Cf~ r.~;.-..

E~ numb~
P~r ~ d~ r-; 1 2 3 Virus souroe DAlS-gal DAB-gal H~VND7 DAB-gal H~VND7 Titer of norn~ I auvest 4.4 x 10 6 2.1 x 10 6 3~2 x 10 5 5x106 SxlO5 Titer of virus ~r- 6X108 7.4x107 3.2x107 2.9x108 3.9x107 Star~ngvoluune 80 n~ .39 n~ 39 n~ 118n~ 40n~
Final --- volurne .S n~ .36 n~ .36 n~ .78n~ .28n~
Fold virus . 136X 34X lOOX S8X 78X
vi}us reoovery 87% 30% 91% 50% 99~/0 As is evident from Table 7, virus rcCov~ly ranged from 30~/0 to 99%, ~,vith the best recovery being obt~ine~l from human producer cells (HX/ND7; recovery ranged from 91%
to 99%).

CONCENTRATION OF RECOMBINANT RE~OVIRUSES BY ULn~

S-500 pl~rifiPd s~p~ co~ p the ,B-gal CA~ ingreco-~ Jvhus DX/CB-bgal and partially ~lu~ ed :!iu~ cr~ inf~. the same virus were each filtered through a 0.45 um filter, and loaded into a CENI~RIPREP-100 filter (product #4308, Amicon, MA). The :iUpellla~lllS were kept at a telllpel~ re of 4~C throughout this procedure, in~lutling during centrifilg~ti~n The CENI~IPREP filters were spun three times each for 45 to 60 mimltPS at 500 x G. Between each spin the filtrate was ~ec~nte~l The retçnt~te was thus sequentially reduced, such that the initial 15 ml (or 10 ml) volume was reduced to a~pl ~ l ely 0.6 ml per unit.
The rPsl-lt~nt titer was determined by assaying HT1080 target cells set up at a cQnc~ntration of 1 x 105 cells per well 24 hours prior to tr~n~ ctiQn ofthe viral sample.

w 096/33282 PCTrUS96/05638 Cells were tr~n~ ced in the presence of 8 ~lg/ml polybrene and 2 mL growth media(DMEM plus 10% FBS) per well. As shown in Table 8 below, app.~ ly one hu-~cd percent ofthe virus was ~ccu~c cd uti~ lg this procedure (note that titers are in BCFU/ml).
S
Table 8 Pre~ ~ Final S-500 4X107/15n~ 1.3X109/0.6 part. COnG 3 x 108/10 ml 1 X 101~/0.6 ml PREPARATION OF~F~Rn~ANT RETROVIRUS ]N A BIOREACTOR

A. Free~i"g protocol Producer cells are frozen in DMEM media co~ g 10% to 20% FBS, and S to 15% DMSO, at a co~ ion of 1 x 107 cells/ml/vial. Cells are frozen i'n a controlled rate freezer (Series PC, Controlled Rate Freezing System, Custom Biogenic Systems, Warren MI) at a rate of from 1 to 10~C per minute. Frozen cells are stored in liquid 20 nitrogen.

B. Bioreactor protocol Cells are thawed from frozen vials at 37~C, washed once with media to remove DMSO, and Pyp~n~led into 850 cm2 nFALCON" roller bottles (Corning, Corning, NY) 25 Pxp~nrled cell culture is used to inoC~ te a RCELLIGEN PLUS" bioreactor (S liter wo~king volume; New Brunswick, Edison, NJ). The cells are grown on microcarriers (i.e., Cytodex 1 or Cytodex 2; Pharmacia, PiscaLaw~y, N.J.) at a concentration of 3 to 15 g/L
microcarrier. Initial inoc~ tiQn d~n~ities are from 4 to 9 cells/bead at halfto full volume for 2 to 24 hours. The media cQn~ ent~ for virus prod~lcti~n are DMEM-high glucose 30 (Irvine ~ri~ntifir; Santa Ana, CA.) basal media supp~ ecl with FBS (10 to 20%), CTlllt~minf~. (8 to 15mM), glucose (4.5 to 6.5 g/L), Non~oss~nti~l amino acids (1X), RPMI
1640 amino acids ( 0.2 to 9.6X), 10 mM HEPES, RP~ 1640 Vitamins (0.2 to 5X).
During culture, pH (6.9 to 7.6) and dissolved oxygen ("DO" 5 to 90%) are controlled by the use of a four gas system which inr1.-des air, oxygen, nitrogen, and carbon W 096/33282 PCTrUS96105638 Yide After several days of batch growth the culture is then cn..~ u,~sly perfilsed with fresh media with concullclll contin-)Qlls harvesting in an esc~ tinf~ pP~il~;~n rate of 0.5 to 2.5 volumes/day. Cell rePntion is the result of dirrele.-lial se~ on of ceD cuv~lcd beads in a dec~ g cQl~lmn S During operation the bioreactor is .. ~ rcd for viable ceDs, titer, ~IIlcosP, lactate, ~mmnni~ levels, andlackofco..l~...;..~l;nn ViableceDsandtiterrangefrom 1 x 105 ceDs/ml to 1 ~10 cells/ml. ( rlllcose ranges from 6 to 0.25 g~, T ~ct~te from I to 25 mM, and ~...i--~n;~ ranges from 0.5 to 30 mM. CeDs are inrub~tPd in the bioreactor for S to 25 days.

* * *

While the present invention has been described above both generaDy and in terms of plerclled embo~iirn~nt~ it is understood that variations and mo~lifir~tiQn~ will occur to 15 those skiDed in the art in light ofthe description, supra Thelt;rore, it is intended that the appended claims cover aD such variations coming within the scope ofthe invention as r.l~imP,d litinn~lly, the p~1hlir~tirJn~ and other materials cited to i~ n;~-le the bacLglou-ld of the invention, and in particular, to provide ~d~1itic~n~l details c~l~c~ g its practice as 20 described in the clet~iled description and ;i . 'es, are hereby incoll.ul~LIed by lerelellce in their entirety.

CA 022l687l l997-l0-l7 W O 96133282 PCTrUS96/05638 SE QlnE N C E LISl~ N G
(1) rFN~AT lNrORhATION:
(i) APPLICANTS: CHIORN VIAGENE, INC.
(ii) TITLE OF lNv~,..ION: High Efficiency Ex Vivo Transduction of Cells by High Titer Recombinant Retroviral Preparations (iii) NUM8ER OF ~yur;N~r;S: 10 ( iv ) C~R k ~: C~ sNcE AnD-RT~!~es s IA AnDR~C~cT~T! Chiron Viagene, Inc.
,BI STREET: 4560 Horton Street CJ CITY: r - yvill~
~DI STATE: California EJ ~Uh~Y: U.S.A.
~F~ ZIP: 94608 (v) COMPUTER RTAnARTT.! FORM:
,Aj MEDIUM TYPE: Floppy disk ,8, COMPUTER: IBM PC - -t~hle ,C, OPERATING SYSTEM: PC DOS/MS-DOS
~DJ SOFTWARE: P~tentTn R~le~e ~1.0, Ver~ion ~1.25 (vi) ~uKkh~ APPLICATION DATA:
(A) APPLICATION NUM8ER: Una~aigned (B) FILING DATE: 19 April 1996 (C) CLASSIFICATION:
(viii) A..u~Nr;~/AGENT INFORMATION:
(A) NAME: Rruse, N -n J.
(B) REGISTRATION NUMBER: 35,235 (C) k~rr;K~N~r;/DOCKET NUMBER: 1157.100 ( iX ) TF~T FCnMMTTNICATION lNr OR~ATIoN:
(A) TELEPHONE: (510) 601-3520 (B) TELEFAX: (510) 6S5-3542 (2) lNruK~ATION FOR SEQ ID NO:l:
(i) ~r;uur;Nur; CHARACTERISTICS:
Al LENGTH: 21 ba~e pairs Bl TYPE: nucleic acid ~C, s~RpNnT.!nN~c5: 8ingle ~D~ TOPOLOGY: linear ( ii ) MnT~T.!CUT~T~ TYPE: cDNA
(xi) ~r;yur;Nur; DTCrRTPTIoN: SEQ ID NO:1:
TAATAAATA~ ATTTAGATTT A 21 (2) INFORMATION FOR SEQ ID NO:2:
(i) ~l!;UUl!iN~;r; C-~ARA~T~RT~CTICS
~A, LENGTH: 35 base pairs IB TYPE: nucleic acid ,C, STR~NDEDNESS: ~ingle ~Dl TOPOLOGY: linear ( ii ) ~nT~CuT~ TYPE: cDNA
(xi) ~r;yU~N~r; DESCRIPTION: SEQ ID NO:2:
GCCTCGAGAC GATGAAATA~ ACAAGTTATA TCTTG 35 W 096/33282 PCTrUS96/05638 ( 2 ) lNr Ok~SATION FOR SEQ ID NO: 3 s ($) SEQUENCE rTTARAcTT2RTcTIcs A J LENGTH: 3 5 ba~e pair~
B TYPE: nucleic acid C STRA~TnT~nNF~.cs 8ingle - ~ D J TOPOLOGY: linear (ii) MnT-lZc~TT~ TYPE: cDNA
(xi) ~I~:yu~ T'SrRTPTION: SEQ ID NO:3:
GAATCGATCC ATTACTGGGA ~G~ CGA CCTGG 35 (2) INFORMATION FOR SEQ ID NOs4:
(i) ~JSyUL_. SE CTTARArT~!RTCTICS:
.'A'I LENGTH: 40 ba~e pairs 8, TYPE : nucleic acid ,C, STRA~ -SS: 8ingle " D ~ TOPOLOGY: linear ( ii ) MnT~T!crJT ~! TYPE: cDNA
(xi) ST2QUr~- ~ DTZcr-~RTpTIoN SEQ ID NO:4:
GCCTCGAGCT CGAGCGATGA AA~rAq~ArAA~ TTATATCTTG 40 ( 2 ) INFORMATION FOR SEQ ID NOs 5 s ( i ) ~;yu ~ c. CHARACTERISTICS:
,'A'I LENGTH: 27 base pairs B, TYPE: nucleic acid C J STRA~IDEDNESS: single ~ D ~ TOPOLOGY: linear ( ii ) MnT.FCUT T! TYPE: cDNA
(xi) ::iJSyU~;N~;~; DESCRIPTION: SEQ ID NO:5:
GTCA~ TGCTATT 27 ( 2 ) INFORMATION FOR SEQ ID NOs6:
( i ) ~iy~ ~ ~ CHZ~RACTERISTICS:
A J LENGTH: 2 7 ba~e pairs , B, TYPE: nucleic acid rC I STR~NL I~N~;SS: ningle ,,DJ TOPOLOGY: linear ( ii ) MnT T~!CUT T2 TYPE: cDNA
(xi) ~l~yU~NC;I~ DT2SrRTPTION: SEQ ID No:6:

AATAGCAACA AAAArAAA~G AGATGAC 27 ( 2 ) INFORMATION FOR SEQ ID NO: 7:
( i ) xwyu ~ wc:~: CHARACTERISTICS:
~AJ LENGTH: 40 ba~e pair~
BJ TYPE: nucleic acid C I STR~NDEDNESS: single DJ TOPOLOGY: linear CA 022l687l l997-l0-l7 W 096/33282 PCTrUS96/05638 ( ii ) MnT~FCUT~ TYPE: cDNA
(xi) ~yur:NCE D~-CrRTPTION: SEQ ID NO:7:

(2) INrOk~ATION FOR SEQ ID NO:8:
(i) ~yu~NCE CHARACTERISTICS:
~A LENGTH: 21 base pairs ~BJ TYPE: nucleic acid C sTRANn~nN~SS: ~ingle ~DJ TOPOLOGY: linoar ( ii ) ~ ~~Cr~T-~ TYPE: cDNA
(xi) ~:yu~ : D~SCRTPTION: SEQ ID NO:8:
ATAAATArAA GGCCTGATAT G 21 (2) lNrORhATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
~A LENGTH: 35 baue pairs ~BI TYPE: n~cle;c acid Cl STRANn~nN~CS: single ~D~ TOPOLOGY: linear ( ii ) ~nT ~CUT~ TYPE: cDNA
(xi) ~yurr~ D~C~RTPTIoN: SEQ ID NO:9:

(2) INFORMATION FOR SEQ ID NO:10:
(i) ~yu~N~ CHARACTERISTICS:
IA LENGTH: 35 base pairs ~B~ TYPE: nucleic acid C~ STRANn~nN~CS: single ~DJ TOPOLOGY: linear ( ii ) ~nT-~CuT-~ TYPE: cDNA
(xi) ~:yu~N~ D~SrRTPTION: SEQ ID NO:10:

Claims (34)

WE CLAIM:

1. A method of producing transduced mammalian T cells or non-dividing cells, themethod comprising:
(a) obtaining a population of T cells or non-dividing cells from a patient; and (b) transducing the population of T cells or non-dividing cells ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest.

2. The method of Claim 1 wherein said T cells are isolated CD4+ T cells.

3. The method of Claim 1 wherein said T cells are isolated CD8+ T cells.

4. The method of Claim 1 wherein the gene of interest encodes a protein or active portion of a protein selected from the group consisting of a cytokine, a colony stimulating factor, a clotting factor, and a hormone.

5. The method of Claim 4 wherein said clotting factor is factor VIII.

6. The method of Claim 1 wherein the patient is a human suffering from a diseaseselected from the group consisting of a genetic disease, a cancer, an infectious disease, an autoimmune disease, a cardiovascular disease, degenerative disease, and an inflammatory disease.

7. A composition comprising an isolated population of mammalian T cells or non-dividing cells, transduced ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant particles carry a vector construct encoding a gene of interest.

8. The composition of Claim 7 wherein said T cells are isolated CD4+ T cells.

9. The composition of Claim 7 wherein said T cells are isolated CD8+ T cells.

10. The composition of Claim 7 wherein the gene of interest encodes a protein oractive portion of a protein selected from the group consisting of a cytokine, a colony stimulating factor, a clotting factor, and a hormone.

11. The composition of Claim 10 wherein said clotting factor is factor VIII.

12. The composition of Claim 7 wherein said mammalian cells are human cells.

13. A mammalian T cell or non-dividing cell transduced ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest.

14. The T cell of Claim 13 wherein said T cell is from an isolated population of CD4+ T
cells.

15. The T cell of claim 13 wherein said T cell is from an isolated population of CD8+ T
cells.

16. The T cell or non-dividing cell of Claim 13 wherein the gene of interest encodes a protein or active portion of a protein selected from the group consisting of a cytokine, a colony stimulating factor, a clotting factor, and a hormone.

17. The T cell or non-dividing cell of Claim 16 wherein the clotting factor is factor VIII.

18. A method of treating a patient having a genetic disease, the method comprising:
(a) obtaining a population of T cells or non-dividing cells from the patient;
(b) transducing the population of T cells or non-dividing cells ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the genetic disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced T cells or non-dividing cells.

19. The method of claim 18 wherein said cell population is a T cell population, wherein said disease is ADA deficiency, and wherein said gene of interest is ADA.

20. The method of Claim 18 further comprising expanding the transduced population of T cells, non-dividing cells prior to re-introduction of the cells into the patient.

21. A method of treating a patient having cancer, the method comprising:
(a) obtaining a population of T cells or non-dividing cells from the patient;
(b) transducing the population of T cells or non-dividing cells ex vivo with a preparations of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating cancer; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced T cells or non-dividing cells.

22. A method of treating a patient having an infectious disease, the method comprising:
(a) obtaining a population of T cells or non-dividing cells from the patient;
(b) transducing the population of T cells or non-dividing cells ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the infectious disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced T cells or non-dividing cells.

23. The method of claim 22 wherein said cell population is a T cell population, wherein said infectious disease is AIDS, and wherein said gene of interest encodes a mutant HIV
protein.

24. The method of claim 22 wherein said cell population is a T cell population, wherein said infectious disease is AIDS, and wherein said gene of interest encodes a ribozyme.

25. The method of claim 22 wherein said cell population is a T cell population, wherein said infectious disease is AIDS, and wherein said gene of interest encodes a synthetic or naturally occurring T cell receptor.

26. A method of treating a patient having an inflammatory disease, the method comprising:
(a) obtaining a population of T cells or non-dividing cells from the patient;
(b) transducing the population of T cells or non-dividing cells ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the inflammatory disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced T cells or non-dividing cells.

27. A method of treating a patient having a degenerative disease, the method comprising:
(a) obtaining a population of T cells or non-dividing cells from the patient;
(b) transducing the population of T cells or non-dividing cells ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the inflammatory disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced T cells or non-dividing cells.

28. A method of treating a patient having a cardiovascular disease, the method comprising:
(a) obtaining a population of T cells or non-dividing cells from the patient;
(b) transducing the population of T cells or non-dividing cells ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the cardiovascular disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced T cells or non-dividing cells.

29. The method of claim 26 wherein said cell population is a T cell population, wherein said cardiovascular disease is hyperlipidemia, and wherein said gene of interest encodes apolipoprotein E.

30. A method of treating a patient having an autoimmune disease, the method comprising:
(a) obtaining a population of T cells or non-dividing cells from the patient;
(b) transducing the population of T cells or non-dividing cells ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a gene of interest useful in treating the autoimmune disease; and (c) re-introducing into the patient a therapeutically effective amount of the population of transduced T cells or non-dividing cells.

31. A method of modulating the activity of a population of T cells or non-dividing cells in a patient comprising:
(a) obtaining the population of T cells or non-dividing cells from the patient;
(b) transducing said population of cells ex vivo with a preparation of high titer recombinant retroviral particles substantially free from contamination with replication competent retrovirus, wherein the recombinant retroviral particles carry a vector construct encoding a protein capable of activating a prodrug;
(c) re-introducing said population of cells into the patient; and (c) administering said prodrug to said patient.

32. The method of claim 31 wherein said protein is thymidine kinase.

33. A method according to Claim 1 wherein an envelope protein of the high titer recombinant retroviral particles is an envelope protein derived from a type C retrovirus or from a type D retrovirus.

34. A method according to Claim 1 wherein an envelope protein of the high titer recombinant retroviral particles is an envelope protein is selected from the group consisting of a retroviral amphotropic envelope protein, a retroviral ecotropic envelope protein, a retroviral polytropic envelope protein, a retroviral xenotropic envelope protein, a gibbon ape leukemia virus envelope protein, and a VSV-g protein.