CA2249987A1 - Packaging cell line based on human 293 cells - Google Patents

Abstract

The invention features a cell line harboring a first expression construct encoding functional Friend Murine Leukemia Virus gag/pol. In a preferred embodiment the cell line harbors a second expression construct encoding a functional viral envelope protein. In more preferred embodiments the viral envelope protein is a retroviral envelope protein; the viral envelope protein is a non-retroviral envelope protein; the cells of the cell line are stably transfected with the second expression construct; the cells of the cell line are transiently transfected with the second expression construct; and the cells of the cell line are stably transfected with the first expression construct and said second expression construct.

Description

WO 97/35996 PCT/I~ 9 PACKAGING CELL LINE BAS~D ON HUMAN 293 CELLS

Background of the Invention The field of the inventiol1 is packagil1g cell lh1es for the productiol1 of replication-deficient retrovirus and the use of these cell lines h1 somatic ceil therapy.
Retrovirus are virus in which genomic RNA is enclosed in a viral particle that is enveloped by a lipid bilayer. Durhlg viral replication the retroviral RNA is reverse 10 tr.lnsclibed into DNA (provirus) by viral reverse transcriptase. The DNA can then integrate into cellular genomic DNA.
Because retroviral vectors can mediate the efficient transfer of genes into the gel1ome of a wide variety of cell types, retroviral vectors have long been considered a promising tool for gene therapy. For safety reasons it is important to use a defective l S retl-ovirus which cannot replicate outside of the targeted cell or beyond the patient to whom it is administered. A replication deficient retrovirus is created by eliminating all or part of one or more of the retroviral structural genes g~lg, pol and etlv. Unlike a normal retrovirus, which will produce infectious virion upon its introduction into a cell, a replication-deficient retrovirus will not produce infectious virion unless the cell provides ~0 the missing viral structural proteins. A cell line which provides the required structural proteh1s is referred to as a packaging cell line.
The use of replication deficient retrovirus does no~ completely eliminate the ri~l~s associated with the use of retrovirus. This is because retroviral DNA can recombine with helper genes present in the packaging cell line or with retroviral elements that are '~5 endocenous to the cellular genome, leading to the production of replication competent rctroYilus. Packaging cell lines employing helper genes with reduced homology to the retloviral vector with which they are used have been developed in an effort to reduce the potential for recombinatiol1. To further reduce the opportunity for recombination, p.lcli;lginc cell lines in which the ~ o~ (71 and e)ll~ gel1es are sepal-alely h1legrated into the 30 p.acli.lgil1g cell gcnomc have been developed.

SUBSTI~I~TE SHEE~ (RU~E 26) Wo 97/35996 PCT~96/00439 Miller (~ona)l Gelle T~lerap!~ J:5, 1990) provides a review of retrovil-us cell packa;,ing lines.
Danos et al. (Proc. N~l~l. Aca~l.. Sci. USA 85:6460,1988; W O 90/02806) desclibe packaging cell lines based on NIH 3T3 cells. These cell lines harbor mutant S val-iallts of the Moloney murine leukemia virus gab~ ol and e~ genes which were intloduced by sequential transfection. Both the ,~ ,J/1)ol and ~ genes employed by Danos et al. Iack functional psi packaging sequences.
Markowitz et al . ( Vil olo~ 167:400, 198~) describe a packaging cel l l ine in which the ~lg/pol and ellv genes are on separate plasmids. The g~ /pol gene used in this 10 cell line was derived from Moloney murine leukemia virus, and the ellv gene used was derived from the amphotropic 4070A env. Both genes were introduced into NIH 3T3 cells. Malkowitz et al. (J. Virol. 62: 1120, 19~8) describes similar eco~ropic packaging cell lines.
Jolly et al. (WO 92/05266) describes packaging cell lines expressing the I S 4070A envelope.
Imler et al. (WO 94/28142) describe replication deficient adenoviral vectors.
Finer et al. (WO 94/29438) describe packaging cell lines basecl on 293 cells transiently transfected with retroviral helper sequences.
There is a need for new, stable packaging cell lines that permit the production ''O of high titers of retrovirus vectors in large-scale cultures. There is also a need for safer pacliaging cell lines.
Summary of the Invention The invention features a cell line harboring a first expression construct encoding functional Friend Murine Leukemia Virus gag/pol. In a preferred embodhnent the cell line harbors a second expression construct encoding a functional viral envelope pl-otein. An expression construct encoding a functional gag/pol is an expression constmc~
which can express gag and pol that is capable of fulfilling the functional role of these proleill.~. hl the viral life cycle (e.g., is capable of acting with env and an appropriate SUBSTITUTE SIJEET (RULE 26) , . _ . _ _ , . . . .

WO 97/35996 PCT/IL ~~ ~9 retroviral vector to produce virion). Likewise an expression construct encoding a fullctional envelope is an expressioll construct which encodes an envelope protein which can flllfill the functional role of the envelope protein in the retroviral life cycle (e.g. is capable of acting witll gag and pol and an appropriate retroviral vector lo produce Vil iOIl).
5 A cell line ks a clonal cell pop~llation generally of imlnol-talized cells.
In mol-e preferled embodilllents the viral envelope protein is a retlovilal envelope protein; the viral envelope proteill is a non-retroviral envelope protein; the cells of the cell line are stably trallsfected with the second expressioll construct; the cells of the cell line are transientl~ transfected with the second expression construct; and the cells of 10 the cell line are stably transfected with the first expression construct said second expression construct.
In an even more preferred embodiment the first expression construct is integl-ated into the genome of the cells of the cell line at a first location and the second expression construct is inserted into the genome of the cells of the cell line a second 5 location distinct from the first location.
In other preferred embodiments the cells of the cell line are transiently transfected with said first expression construct; the cells of the cell line are stably tr;lllsfected with said first expression construct; and the Friend Murine Leukemia Virus is ~strain FB29.
In still other preferred embodiments the envelope is an amphotropic envelope;
alld the envelope is an ecotropic envelope.
In another embodiment the invention features a cell line produced by tr;lnsfecting human 293 cells with the first expression constmct to obtain a gag/pol expressing cell ~ine and then transfectinp the gag/pol expressing cell line with the second 25 expressioll construct. In a more preferred embodiment the cells are stably transfected with the fin~it and second expression constructs.
In another embodiment the invention feature~s a cell line produced by ~r.lll.sfccting human 293 cells with the secolld expression con~struct to obtain an envelope SUBSTITUTE SHEET ~RULE 26) ., . , . _ , W O 97/35996 PCT~B96/00439 expressing cell line and then transfecting said envelope expressing cell line with the first - - expression construct. In a more preferred embodiment the cells are stably transfected with the first and second expression constructs.
In other preferred embodimellts the first alld second expression constrLlcts do 5 not include a functional retroviral LTR.
The cell lines are preferably based on the human cell line 293. In one embodiment the packaging cell line expresses the gag/pol genes of Friend Murine Leukelnia Virus (FmuLV) strain FB29 and the 4070A env gene. ln another preferredembodiment the cell. Iine harboring the first and second expression constructs further 10 halbols a retroviral vector, the cell Ihle being capable of producing virion.In another aspect the invention features virion produced by a cell line cell line harboring a first expression construct encoding functional Friend Murine Leukemia Virus gag/pol, a second expression construct encoding a functional viral envelope protein.
In yet another aspect, the invention features a method for producing virion 15 comprising. The method includes: (a) stably transfecting a cell line harboring the first and second expression constructs with a selected replication-incompetent retroviral vector which comprises a packaging sequence to create a producer cell line; and (b) culturing the producer cell line under conditions causing production of virion.
In preferred embodiments of the method the packaging sequence is not the ~0 Friend Murine Leukemia Virus FB29 psi sequence and the packaging sequence is a retrotlansposon virus-like 30 S RNA p~i sequence.
A retroviral vector is a retroviral nucleic acid deleted for some or all of the sequences encoding viral proteins (gag, pol, and env) and are therefor replication deficient. Retroviral vectors generally include one or more heterologous (non-retroviral) 25 genes. Retroviral vectors are often used to transfer the helerologo~ls gene(s) to cells which the virus infects. e.g., for somatic cell therapy. Retroviral vectors generally include (or retain) sequences necessary for packaging and integration. They also include sequences required for expression of the heterologo~ls genes. In many cases the expression and SUBSTITUTE SHEET (RULE 26) , .. ..

W O 97/3S996 PCT~B96/00439 packaging sequences are not all derived from the same retrovirus. Expression sequences - - from noll-vil al sources are commonly used.
A cell which expresses the viral proteills required to complement a replication deficient retrovirus is referred to as a packaging cell. Packaging cells generally do not 5 include the cis-acting retroviral sequences which actually direct dimerization and enc.lpsulatioll. A packaging cell which harbors a retloviral vector and can produce virion contailling retroviral genetic material, including any inserted heterologous (non-retroviral) sequences, is referred to as a producer cell line. Packaging cell line have uses beyond somatic cell therapy as well.
The packaging cell lines of the invention have a number of advantages. The stability of tlle cell lines is one important advantage. For example, the clone designated ~9~-E 17 is able to stably produce high virus titers even in the absence of any selection for either expression of structural genes or for the retrovirus vector produced by these cells.
Titers are stable for at least 10 cell passages and decrease only by a factor of 5 after ~5 I S cel I passages.
As described briefly below, and in greater detail in the examples, the sequential transfection of first the gag/pol expression vector and then the env expression vector assures that these vectors integrate at separate sites within the host cell genome.
Recombinatioll events which might otherwise occur between plasmids when they are co-~0 transfected are thus excluded. The vectors can also be integrated in the opposite order.
The fact that gas~/pol genes employed hl the packaging cell lines of theinvention are derived from Friend Murine Leukemia virus FB~9 and not from Moloney Murine Leukemia virus has several advantages. Optimizations may have occurred which, taken together, assure high vilUS titers, a higher physical stability of the virus particles or 75 ~reatel halve-lives of the particles. The strength of the promoters, the nature of the stluctural proteins, the kinetics of assembly of gag/pol encoded proteins into nucleocapsids, and the efficiency of maturation and release of virus palticles from the hltecle(l cell, which reqLIile.s inter.lctioll of nucleocapsids with the envelop proteins pre~sent ~UBST~TUTE SHEET (RUEE 26) , W O 97/35996 PCT~B96/00439 in the cellular membrane, can all contribute to the higher titer, greater physical stability - - and longel hal~:life of the vir~l particles produced by thc packaging cell lines of the invelltioll. Moreover, the physical properties of virus particles based on FMuLV may facilit;lte vector purification and vector concentration processes indispensable for the S prod~lction of large quantities of retl-ovil-~ls vector particles for gene therapy purposes eithe r il~ l~ivo or ex vi~o.
AdditionLIl advantages can arise from the sequence dift'erences between FMuLV and MoMuLV. Currently used relrovirus vectors contain a large psi pacl~aging recioll which extends into the gag coding region (e.g., up to the NarI site at position 1035 10 ol' MoMuLV). This psi region thus overlaps with the entire gag sequence encoding matri,Y
protein present in packaging cells. Although the retroviral vectors can be mutated (i.e., the gag ATG codon can be changed to a stop codon), there is at least a 365 bp overlap (between the original ATG and the Narl-fragment) which Call permit unwanted recombination. Such an event, of course, would not give rise to replication competent 15 viruses. However, it would create a product which could be subject of furtherrecombination events and could be co-encapsulated into virus particles. The lower homology of only 83.6% (68 mutations in 416 bp) between FMuLV and MoMuLV in thisregioll could help reduce the frequency of recombination. All homology can be abolished by replacing the MoMuLV packagillg sequence with a VL30 packaging sequence (see 20 WO/960 1 324).
Brief Description of tlle DrawinOs Fig. I is a schematic drawing of the construction of the pTG5332 vector containing a FB29 FmuLV gag/pol gene. Fig. IA is a schematic drawing of the constluction of the pTG533 I vector from FB29 FmuLV gag/pol gene and p poly III. Fig.
25 I B is a schematic drawing of the construction of the pTG5323 vector from FmuLV
gag /pol gene and p poly IIl and M 1 3TG30. Fig IC is a schematic drawing of theCOllStl'UCtiOIl of the pTG5332 vector from pTG533 1 and pTG5323 vectors.
Fig. 2 is a schematic dr.lwing of thc constr-lction of thc pTG5349 vector RECTIFIED SHEET(RULE 91) ISA/EP

W O 97/35996 PCT~B96/00439 capable of expressing a FB29 FMuLV gag/pol gene. Fig. 2A is a schematic drawing of - - the construction of the pTG5324 vector from p DELTAE, pBCMGNeo and p poly III
vectors. Fig. 2B is a schematic drawing of the construction of tlle pTG5354 vector from pY3, pTG6529 and p poly III vectors. Fig. 2C is a schematic drawillg of the construction 5 of the pTG5349 vector from pTG5345, pTG5332 and pTC5334 vectors.
- Fig. 3 is a schematic drawing of the constluctioll of the pTG5387 vector capable of expressing the amphotropic env gene of 4070A. Fig. 3A is a schematic drawillg of the construction of the pTG5347 vector from pBCMGNeo, p DELTAE and ppoly III. Fig 3B is a schematic drawing of the construction of the pTG5383 vector from 10 pPAM3 and pBSK+ vectors. Fig. 3C is a schematic drawing of the construction of the pTG5387 vector from pTG5347 and pTG5383.
Fig. 4 is a schematic drawing of the construction of the pTG5366 vector capable of expressing the FB29 FMuLV ecotropic env gene.
Fig. 5 is a schematic drawing of several retroviral vectors.
Fig. 6 is a flow chart of the construction of a stable amphotropic packaging cell line based on 293 cells.
- 400 clones tested for reverse transcriptase (RT) activity.
- 28 clones with highest RT activity were transfected with amphotropic envelop pTG5387 and retrovirus construct pTG5391 (FB29 LTR - LacZ - SV40-Puro).
'~0 - Transient titers and titers of mycophenolic acid/puromycin selected pools were determined.
- After selection, only few clones continuously produced high virus titers and grew well.
- 30 subclones were tested for RTase activity.
~5 - The subclones were transfected with pTG5391 and pTG5387 to test for productivity.
- Transient vir us titers and titers of puromycin selected pools were determined.
- All clones were tested for productivity by trallsiellt and st.!ble transfection RECTIFIED SHEET (RULE 91 ) ISA/EP

W O 97/35996 PCT~B96/00439 with pTG5391.
- Subclones of 275- 12 produce only in transient assays higher titers than subclones of 231-6.
- Subclones of 231-6 grow better alld produce higher titers after puromycin selection.
- The best 40 clones were retested by transient tr~lnsfection with pTG5391 and titration.
- The best eigllt clones, 2.9, 2.6, 21.24, 28.10, 4.8, 17.6, 17.16, and i 8.4 were renamedA,B,C,D,E,F,G,H,respectively.
10 - 133 subclones were tested for productivity by transiellt and stable tr~lllsfection of pTG5391.
- Most subclones of A, B, D, F, G, H grow slowy, contain giant cells and produce low titers. They do not support the subcloning procedure by limited dilution.
15 - Almost all subclones of C and E produce high titers and grow well.- Clones E 17 and C 18 grow the best alld start off the fastet after thawing.
- Clones E are very sensitive tow~rds puromycin ( l lug) but grow fast to confluency.
- Clones E support best the subcloning procedure after transfection of retrovirus constructs.
- St~ble transfection of pTG5391 into E17 and E20 rcsults in titers of 5xlO E6.
Selected subclones E 17-5391 and E20-5391 produce up to Sx IOE7 cfu/ml.
- Stable transfection of pTG9325 into E 17 results in a titer of 4x 10 E6.
(Individual clones of PA317-9325 cells produce only IxlO E6).
Selected stable subclones E 17-9325 and E20-9325 are being titered.
- Serial passages and titrations with subclones E 17-5391 and PA317-5391 ~rc being performed to tesl for long-term stability.

RECTIFIED SHE~T (RU~ ~ 91 ) ISA/EP

Fig. 7 is a photograph of the results of an experiment performed to assess the transduction ability of virus produced by a packaging cell line of the invention.
Fig. 8 is a schematic drawing of a retroviral vector which can be used in conjunction with the packaging cells of the invention to create a producer cell line.
S De~scription or the Prefelled Embodin~ellt~s Described below are retrovh-al packagillg cell lines useful for the production of high titer retrovirus vectors. The packaging cells of the invention are based on human cells or other cells have very few or no endogenous retroviral sequences (e.g., human 293 cells).
Using human cells as packaging cells decreases the risk of producing replication competent retroviruses by eliminating endogenou~s murine retroviruses. The use of human packaging cells also avoids the problem of serum inactivation of virus vector particles~ Human 293 cells (Graham et al., J. Ce/l. Virol. 36:59, 1977) have been stlown to be useful for high titer retrovirus expression. These cells contain the Adenovirus 15 5 E I A and EIB region, the gene products of which transactivate transcription of transfected Oenes. This meets the requirements for high level production of viral .structural proteins and RNA.
In addition to producing virion for use in somatic cell therapy, producer cells can be directly injected into a patient. Direct injection has been shown to be more ~0 efficient than injection of virion (Tamiya et al., Ge~2e Tllerap~! 2:531, 1995).
In one embodiment, the packaging cells of the invenlion also harbor the Adenovirus E I region (Graham et al., J. Gen. Virol. 36:59, 1977) and thus encode proteins which transcriptional activators for a large number of eukaryotic promoters.
These cells can support high level expression of retroviral structural genes that are under S the control of a non-retroviral promoter.
The retroviral structural genes expressed by the packaging cell lines of the invelltion are divided between two expression plasmids. Tllis reduces thc risk of rccolnbin;ltion between these .structur;ll gene~ and ally retrovil-ui vcctor amplified in the.~c SUBSTITUTE S~IEET (RULE 26) cell lines. Moreover, the retroviral structural gene encoding plasmids lack known retlovilal regulatory se~uences.
The pacl;aging cell lines described below have a numbel ot useful features which ale more readily undelstood by reference to the mal1llel in which they were 5 prod~lced.
First, the vectors used to express retrovil-L11 structural proteins prefcrably employ non-retroviral control elements. In one embodiment, the invelltion features gag/pol and env expression constructs employillg a cytomeg,llovilus promoter, rabbit beta globin intron 2 intron and poly A sequences, a mouse mitochondrial 12S DNA sequence 10 (useful for introducing several copies of the expression plasmid into actively transcribed genome regions), and a selection expression cassette which includes the SV40 promoter, the SV~0 poly A sequence, and a gene conterring drug resistance. This last feature permit.s dh-ect selection of cell clones which, after transfection, have stably integrated lhe plasmids into their genome. This avoids the need to co-transfect a plasmid carrying a 15 selective marker along with the expression vector.
In some embodiments, 18bp of retroviral sequcnce upstream of the gag/pol ATG and 7 bp of retroviral sequence upstream of the e~lv ATG remain. ~n some cases it may be desirable to remove all retroviral sequences. Finer et al. (WO 94/29438) describe all approach which permits removal of all retroviral sequences upstream of the gag/pol 20 alld el~v coding sequences.
The cell lines of the invention are prepared using a two step transfection in which the expression construc~ encoding gag/pol and the expression construct encoding env are separately transfected. This two step process reduces the opportunity for unwanted recombination events. One can, of course, transfect with either vector first.

2~ In the example described below, the packaging cell lines were produced by first transfectillg 293 cells with a vector which encodes the FMuLV FB29 gag/pol genes.
Positive selection with hy,,romycin B pcrmitted identification of transfected clones.
Stably trallst'ected cells wcre tnen transt'cctcd with ~I vcctor cxprcssillg all amphotropic SUEISTITUTE SHEET (RUL' 261 , . . .

WO 97/35996 PCT/Is96/00439 -gene. When this cell line was then transfected wittl a retroviral vector, virion having a broad host range can be produced. Virion produced by this packaging cell line are able to efficiently transduce human, canine, feline and mouse cells. These viral particles may also be used to hlfect all cell types which are hlfectable by thc amphotlopic 4070A Vil-US.
5 Alterllatively, the stable gag/pol expressillg cell lhle can be trallsfected with a vector capable of expressillg the ecotropic FMuLV FB29 e~ gelle. When tllese cells are transtected with a retloviral vector, ecotropic virion can be produced.
The strain FB29 was isolated by cloning viral circLIlal- DNA from cells hlfected with Friend Murine leukemia virus FMuLV I-5 (Sitbon, et al., Cell 47:851, 1986;
10 Mathieu-Mah~ll et al., Virolo~ 119:59, 1982; Perryman et al., Nl~cl~ieAcids Res., 19:69S0, 1991). The clone FB29 proved to be highly virulent in mice (Sitbon, et al., C~ll 47: 851, 1986; Portis et al ., J. vi/ ol. 65: 1877, 1991).
The g~7g/pol genes of the FB29 FMuLV are encoded by over~apping reading frames which cover nucleotides 619-2235 and 2221 -5835 of the retroviral genome 15 (according to the nucleotide sequence disclosed in GeneBank/EMBL under accession number Z 11128) and are generated from the viral mRNA by ribosomal frameshifting.
The FB29 env protein which is of ecotropic type is transcribed from a post-trallscliptionally modified spliced viral mRNA. The 5' coding regioll of the ellv gene overlaps with the 3' end of the pol gene (position 5775-5835).
~0 Perryman et al. (l~l~cleic Acids Kes. 19:6950, 1991) report the complete nucleotide sequence of Friend murine leukemia virus, strain FB29.
Plasmid constructioll To minimize the risk of generating recombinant, replication competent viruses, the gag/pol genes and the ertv gene are expressed from two different vectors. In 25 the example below, the viral structural genes, gag/pol and e~lv, are expressed under control of the Cytomegalovirus immediate early promoter and enhancer (Boshart et al., C~ll 41 :521, 1985). To increase long mRNA half-life and high translation efficiency, the ~ectori used to express retroviral structural genes in the example desclibed below contain SUBSTITUT~ SHEET (RULE 26) W O 97/35996 PCT~B96/00439 the intron and 2 polyA fragment of the rabbit betal-globin gene. The vectors also contain a 1.1 kb fragment of mouse mitochondrial 12 S rDNA from pDelta. This sequence includes elements which cause the formation of he~d-to-tail multimelic stmctures when they integrate into mammalian host chromosomes (Lutfalla et al., So~ tic Cell a~7d '~ Molec~ r Genetics, 11 :223, 1985). Both vector~ inclucie a selectable marker for identificatioll of stably transfected cells. The vector expressing ~,~a~,~//701 includes a hygromycin B resistance gene located between SV40 early prollloter and the SV40 polyA
regioll. The vector expressing ellv includes a mycophenolic acid resistance gene located between SV40 early promoter and the SV40 polyA region.
It should be understood that various of these elements can be replacecl by functiollally similar elements. Thus, the Cytomegalovirus promo~er could be replaced by allothel strong promoter capable of directillg high level constitutive or inducible expression in the chosen cell line. Suitable vectors include, but are not limited to, the BKG promoter, the MT promoter, and the TK promoter. Similarly, the selectable markers 15 can be replace by other selectable markers.
Isolation of DNA, digestion with restriction enzymes, purification, PCR
amplification and other manipulations of DNA fragments used in cloning procedures were performed using methods well known to those in the art of molecular biology. (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold 20 Spring Harbor, NY, USA, 1982). Plasmids were grown on E. coli DH5 alpha (Woodcock et al., Nucleic Acid Res. 17:3469, 1989). Site-directed mutagenesis were performed using tlle Amersham oligonucleotide-directed il1 vitro mutagene~is system (Version 2.1, code RPN 1523) essentially as described in the manufacturer's protocol. The single-stranded M 13 DNA which served as template were purified after phage amplification on NM52 25 cell (Woodcock, Nllcleic Acids ~es. 17:3469, 1988; Gough et al.. J. Mol. Biol., 166:1, 1983) using standard procedures.
Cloning of FB29 FMuLV gag/pol genes: (pTG 5332) This example describes the cloning, from FB29 gCIlOItliC DNA. of two SUBSTITUTE SHEET (RULE 26) W O 97/3S996 PCT/L~r''/~0139 fragments which together include the entire gag/pol gene, the modification of these two fragments by site-directed mutagenesis to introduce new restriction sites, and the reconstit~ltion of the gag/pol coding region to generate vector pTG5332. These plocedures are ill~lstrated schematically in Fig. 1.
First, the Kpnl-EcoRI fragment (n~lcleotides 480-3297) of FB29 was s~lbclol~ed into ttle Kpnl-EcoRI sites of pPolyIlI-I (Lathe et al., Ce/l~ 57: l 93, 1987) resuiting in plasmid pTG5303. USillg the Accl restriction sites in the polylinkel and in the ' gelle (nucleotide 1337), a 881 bp AccI fragment of pTG 5303 was subcloned into the Accl site of M13 mpl30, resulting in M13TG 5306 which was subsequently mutagenized 10 to intl-od-lce a new EcoRI site upstream of gag initiator ATG using OTG54 l 9 (ACAAATACAGGAATTCAGTCAAACAG) (SEQ ID NO.: l ). The 886bp AccI
fr.lglllellt of the resulting vector M 1 3TG 5325, which contains the 5' end of the g~1g gene coding region with the new EcoRI site, was then reintroduced into the AccI sites of pTG
5303 from which the 881 bp AccI fragment had been removed, resulting in pTG 5331.
Second, the HindlII-Accl fragment of FB29 genome (nucleotides 5508-6352) was cloned into the HindllI-Accl sites of M13TG130. Site-directed mutagenesis was perfol-lned on the resulting vector M 13TG 5305 with OTG 5334 (GGCCCCTTTGGTCGACAGGAAGAG) (SEQ ID NO.: 2) to create a new SalI
restl iction site I I bp behind the TAG stop codon of the pol readhlg frame (nucleotide ~0 5~55) giving M13TG5307. This change also serves to minimize sequence homologybetween gag/pol and env expression vectors which could allow homologou~s recombination .
The EcoRI- HindIII fragment of FB29 genome DNA (nucleotides 3297-5508) was introduced into the EcoRI-Hind III sites of pPoly III-I, resulting in pTG5304. The ~5 795 bp Hind III-Sall fragment from M13TG 5307 carrying the 3' end of the pol gene with the new Sall site was cloned into pTG 5304 linearized with HindlII-Sall, resulting in pTG
5323.The entire gag/pol coding region was reassembled by cloning the 2254 bp EcoRI
r.lglllel1t of pTG 5331 into the EcoRHsite of pTG 5323, re~slllting in pTG5332.

SUBSTITUTE SHEET (RUL~ 26) WO 97/35996 PCT/Is96/00439 Construction FB29 FMuLV gag/pol expression vector (pTG 5349) The construction of expression vector pTG 5349 is depicted in Fig. 2.
The Cytomegalovirus immediate early promotor and enhancer region together with the r.1bbit beta- I globin intron-2 polyA region were isolated frolll pBCMG Neo (Karasuyama etal., J.E~l~.Mecl. 169:13, 1989;Kalasuyalllaetal.,E~ J.Iov~ ol. 18:97, 1988)by dicestioll with XbaI, filling in with Klenow fragment of L;: coli DNA polymerase I
("Klenow"), and digestion with BglII. This fragment contains the CMV immediate early promotor and enhancer region from position - 671 to +73, and the rabbit beta- I globhl hltloll-2 polyA fragment from position 903-2063 (GENEBANK accession K03256 and 10 M 12603). The original EcoRI site at position 1542 in the beta- I globin gene had been ch,1nged by introduction of a XhoI linker generating a unique Xhol restriction site into wllich genes of interest can be cloned. This fragment was inserted into pPoly III-I
lhlc.1l ized with XhoI, filled in with Klenow, and then digested with BgllI. The resulthlg plasmid is pTG 5343. A 1.1 kb EcoRI fragment from pTG 5324, filled in with Klenow 15 and containing the mouse mitochondrial 12S rDNA from pDelta ( Lutfalla et al., So/71atic Cell alld Molec~lar Cenetics. 1 1:223, 1985) was then transferred into pTG 5343 linearized with Nael. The resulting plasmid pTG 5345 was then linearized with XhoI and ligated with the XhoI fragment from pTG 5332 which contains the gag/pol genes from FB'9 FMuLV. The resulting plasmid pTG 5348 contains the viral structura~ genes under ~0 control of the CMV promotor but lacks an eukaryote selective marker.
A suitable selectable marker was prepared as follows. First, an SV40 expression cassette was generated from pTG 6529 which is a p polyIII-I plasmid having incol-porated a cassette expressing the puromycin resistant gene (p~c) under the control of SV40 early promoter and SV40 poly A sequence. After digestion with HindIII and partial digestion with XhoI to remove the pac gene. the ends were filled with Klenow and the ~ec~or was religated. The resulting plasmid pTG5353 contains a unique HindIII site l~e(ween the SV40 promotor and poly A regions. The Escllericllia coli Hygromycin B
~-hosphotl-ansferase gene from plasmid pY3 (~Blochlinger et al., Mol. C~ll. Biol., 4:2929.

SUBSTITUTE SHEET /RULE 26~

WO 97/35996 PcT/Isg6/00439 19~4; Gritz et al., Ge~e 25: 179~ 1983) was released as a KpnI-SacI fragment and cloned into pPoly III-I linearized with KpnI and SacI, resulting in plasmid pTG 5352. To gene1ate pTG5354, the SmaI-HilldIII fragment of pTG 5352 containing the hygromycin phosphot1allsfelase gene was filled with Klenow and tlle cloned into pTG5353 linearized with HindIII and filled with Klenow. The XhoI frLI;,ment of pTG 5354 containing SV40 promotol-, hygromycin phosphotrclnsfel-ase gene and SV40 polyA was thell cloned into pTG 5348 linearized with Notl after filling in the ends with Klenow. The resulting plasmid is pTG 5349, the expression vector for FB29 FMuLV ,~ /po1 genes.
Constr~lction of the amphotropic env gene expression vector (pTG5387) The construction of vector pTG5387 expressing the e~ gene is illustrated schematically in Fig. 3.
Plasmid pPAM3 (Miller et al., Mt)l. Cel~ Biol., 6:2895-2902, 1986) contains a SalI-ClaI fragment which includes the ellv gene from amphotropic 4070A virus. The sequence downstream of the ClaI site, which includes the C-terminal 32 amino acids of I ~ the ellv gene, is from ecotropic MoMuLV. The amphotropic ~ItV gene from pPAM 3 (Miller et al., Mol. Cell Biol., 6:2895-2902, 1986) was modified by introducing a new SalI
restriction site 10 bp upstream of the ATG start codon. For this purpose, the XbaI-BamHI
fragment from pPAM3 comprising the 5' region of the amphotropic env gene was cloned into the XbaI-BamHI sites of pBluescript SK+ (Stratagene, La Jolla, CA, USA), resulting ~0 hl pTG 5369. Single stranded plasmid (phagemid) DNA of pTG 5369 was recoveredfrom E. co~i XL I Blue cells infected with helper phage R408 (Stratagene, l a Jolla, CA, USA). essentially as described in the manufacturer's protocol. Mutagenesis of single stranded DNA with oligonucleotide OTG 6057 (CCATGTCCGTCGACAGGATGGTCC) (SEQ ID NO.: 3) was performed to introduce a new Sall site. The resulting plasmid pTG
5380 was linearized with BamHI, and the BamHI fragment from pPAM3 comprising the3' end of the amphotropic env gene was introduced, resulting in pTG 5383. The entire env gene was released by digestion with SalI and cloned into the XhoI site of expression ~ector pTG 5347. Plasmid pTG 5347 was constructed by introducing the Sall-ClaI

SUBSTITUTE Sl IEET (RlJLE 26~

WO 97/35996 PCT~B96/00439 fragment from pDelta (see above) frorn into the NarI site of pTG 5343 (see above) after filling in the end with E. coli DNA polymerase I. The fragment from pDelta includes the mouse mitochondrial 12s DNA and the XGPRT gene, confelring resist.lnce to miclollephronic acid, under control of the SV40 prolllo~ol and the SV40 poly A region.
5 The sequence of the FB29 FMuLV ~ a~ ol gene and the amphotlopic 4070A ellv differ significantly. The amphotropic e~lv and FB29,~ /pC)/ contain 6~ bp that are 90.7C70 homologous.
Clonin" and constl-uctioll of FB29 FMuLV ecotl-opic env gelle expre~ssio vector (pTG5366) The construction of pTG5366 is illustrated schematically in Fig. 4.
The ecotropic FMuLV FB29 env gene (Sitbon~ et al., Cell 47:851-859) was amplified by polymerase chain reaction (PCR) with oligonucleotides OTG 5703 (GAGGATCCATGCATCGGAATCGACATG) (SEQ ID NO.: 4) and OTG 5708 (TAGGATCCATGCATTATTTATTGTGGCTCG) (SEQ ID NO.: 5) using standard 15 protocols. Briefly, 100 ng of tempiate DNA was amplified in a 100 ~I reaction using 100 pmol of primers. By using OTG 5703 and OTG 5708, new NsiI sites were introduced at ~enome positions 5759 and 7809, respectively. The PCR product was cut with Nsil an(l cloned into the PstI site of pPolylII-I, resulting in pTG 5364. Subsequently, the BalI
fragment of the env gene in pTG 5364 was replaced by the identical fragment isolated 20 from pFB29 (position 5991 -7533) to exclude all possibilities of having introduced mutations during the PCR amplification process. The env gene of the resulting plasmid pTG5365 was cut out with Xhol and SalI and cloned into pTG5347. The resulting expression vector pTG 5366 contains ~he entire FMuLV FB29 env gene. The sequenceoverlap between FB29 gag/pol and FB29 env expression vectors can be only 95 bp 25 (position 5762-5857).
Generation of Cell T.ines General technolo~y The 293 cells (Graham et al., J. Cel~ Virol. ~6. 59. 1977) (CRL 157:3, Lol No SUBSTITUTE SHEET (RULE 2 W O 97/35996 PCTnB96/00439 F10150), NIH 3T3 cells (CRL 1685), Mus dunni cells (CRL 2017), canine MDCK cells(CCL 34), feline PG4 cells (CRL 2032), A549 cells (CCL 185), and HeLa cells (CCL 2) were obtained from ATCC (Bethesda, MD). Cells were grown in Dulbecco's modified Eagle'~ medium containing 10% (vol/vol) fetal calf serum, 3 g/ml gl~lcose, 1% non-es.~enti.ll amino acids and 200jug/ml gentamycin. Selection of transfected or infected cells was perfol med with Hygromycin B (Boehringer Mannheim) at 350 ,ug/ml. Selection with mycophenolic acid was performed in medium supplemented with 15 jug/ml Hypoxanthine (Sigma), 15 jug/ml Thymidine (Sigma), 250 ,ug/ml Xanthine (Sigm~l) and 25 jug/mlMycophenolic acid (Serva). Puromycin was used at IIJg/ml.
Plasmid DNA transfection into eukaryotic cells was performed using the calcium phosphate precipitation method of Graham et al. (Virolog!! 52,456, 1973).
Reverse transcriptase activity in cell culture supernatants was determined as described by Goff et al. (J. Vi/ol., 38:239, 1981). ~B-galactosidase activity in cells fixed with formaldehyde/Olutaraldehyde was determined as described by Sanes et al. (EMBO J., 5 5:3133, 1986). Radioactive, 3-P labeled DNA probes were generated using the Amersham multiprime DNA labe~ing system (RPN 1600Y), essentially as described in the mallufacturer's protocol.
To determine retrovirus titers produced from individual cell clone.s expressing either only the FB29 FMuLV gag/pol genes or FB29 FMuLV gag/pol plus amphotropic '~0 env genes, transient transfections were performed in 6 well plates with 40-50% confluent cells, using the retrovirus vectors illustrated in Fig. 5. Vector pTG5363 represents a retlovirus vector carrying the puromycin resistance marker under control of an internal SV40 promotor and the human CD4 cell surface antigen gene under control of the LTR
(LTR-~FB29-hcD4-sv4oprom-pac-LTR) Vector pTG5391 carries the E. coli r~-2~ galactosidase (LacZ) gene under control of the LTR and the puromycin resistance marlcerunder control of an internal SV40 promotor (LTR-'YVL30-LacZ-SV40prom-pac-LTR).
Vector pTG4371 comprises the LacZ gene with a nuclear localization sequence (nls) ciirected by the retroviral LTR (LTR-'PMLV-nlsLacZ-LTR). Fin.llly vector pTG93~5 SUBSTITUTE St~EET (RUL~

WO 97/35996 PcTtIs96/oo439 e,~presses the neomycin-resistant gene (Neo) under the control of the LTR (LTR-~VL30 -Neo-LTR).
The virus particles produced in culture supernatant were then titred on NIH
3T3 cells (ATCC: C'RL 1685) using standald methods. Briefly, Sx 10~ cells in 6 well pl;ltes were incubated for I h hl presence of 15 jug/lnl polybrel1e with 400 ~11 of serial dilutions (10 ~ tolO ') of supernatants from transfecled producer cells culture supernatallts.
After 111, 3 nll of medium was added. Selection for puromycin resistant cells was pert'ormed by replacing the medium 24 h after the infection with 2 ml of DMEM
containing I ,ug/ml of puromycin. The medium was changed every two days. Colonies of 10 puromycin resistant, transduced cells were counted after 10-14 days. Staining for ~-galactosidase activity was performed 48 h after the infection by standard methods.
Transient retroviru~s expression system Five micrograms of each of the plasmids pTG 5349 (FB29 gag/pol) and pTG
5366 ~FB29 env) were transfected together with 15 lug of either vector pTG 5363 (LTR -I ~ CD4-SV40prom-pac-LTR) or vector pTG 4371 (LTR-nlslacZ-LTR) into 293 in 10 cm dishes containing 10 ml DMEM. After 16 h, the medium was changed. Eight hours later the medium was replaced by 4 ml of DMEM. After 24h, the medium was recovered, fillered through 0.451um filters and frozen at -80~C. The virus particles produced in this tl-ansient assay were titered on NIH 3T3 cells ~s described above.
O The results presented in Table I show that titers obtained from transiently trallsfected 293 cells are up to 4-fold higher than titers obtained with stable ecotropic GP+E-86 cell lines (Markowitz et al., J. Virol. 62: 1120- 1124, 1988) ~UBSTITUTE SHEET ~ULE 26) WO 97/35996 PCT~B96/00439 - TABLE I
pTG 4371 pTG 5363 ~ac~) (Puromycin) GP E 86 19 x103 5.5 x103 293 25x103 22 x103 Production of stable gag/pol expressing cell lines Described below is the construction and use of cells stably expressing FB29 gag/pol genes. 293 cells were transfected with pTG 5349 (FB 29 FMuLV gag/pol) using 15 the calcium phosphate precipitation method. Twenty micrograms of DNA were allowed to form precipitates in a volume of 500 ~ll calcium phosphate solution and then added to 10 cm culture dishes containing 10 ml of DMEM culture medium and the target cells at a density of 40-50%. After 16 h the medium was changed. Forty-eight hours after transfection the cells of each dish were split into 5 new 10 cm dishes and selective medium 20 containing 350 ~lg/ml of Hygromycin B was added. The medium was changed ever,v 4 days. After 3 weeks, clones were recovered and cultivated in 24 well plates. When clones became confluent, 24 h-supernatants were recovered from the wells, filtered through 0.45~m filters, and pol expression was determined by reverse-transcriptase activity (RT).
Cell clones with high RT activity were subsequently expanded in 6 well plates, 2s trypsinized and frozen in 10 % DMSO (Sigma), 20% FCS at -80~C.
Table II shows the reverse transcriptase activity of the 293 TG5349 cell clones which, in subsequent experiments, gave the best results. The RT activity in the best clones is 4-fold higher than in the GP+ E-86 cell line, an ecotropic NIH 3T3 based cell line containing the MoMuLV gag/pol genes under control of the MoMuLV LTR
30 (Markowitz et al., J. Virol. 62: 1120- 1124, 1988) .
The 27 clones 293-TG5349 with the highest reverse transcriptase activity were thawed1 grown up and tested for transient virus production in tWO independent SUBSTlTUTt SH'ET (RULE 26) CA 02249987 l998-09-24 WO 97t35996 ~CT/ILr~'00139 experiments. These 27 clones were transfected with 1.5 ~ag pTG 5366 (FB29 env gene) and 3 ,ug pTG 5363 (LTR-hCD4-SV40prom-pac-LTR) in 2 ml DMEM. Titration for puromycin resistant transduced NIH 3T3 cells was performed as described above. As controls, GP+EnvAml2 cells were transiently transfected with pTG5363. In these s experiments, 293-TG5349 cell clone 231 gave 12.5 times higher titers than GP+EnvAml2 cells.

TABLE II

RT activity Puromycin resistant colonies (cpm x 103) Exp.1 Exp.2 mean Titre (x10~) 293-TG5349- 59 27 25 0 6.3 IS 293-TG5349- 82 21 16 0 4.0 293-TG5349- 98 22 4 1 1.3 293-TG5349- 118 16 11 8 4.3 293-TG5349- 122 15 10 11 5.3 293-TG5349- 231 22 80 10 22.5 293-TG5349- 244 16 1 1 0.5 293-TG5349-275 20 90 50 35.0 293-TG5349-280 12 36 20 14.0 293-TG5349-295 12 1 1 0.5 293-TG5349- 298 17 24 18 8.0 2s 293-TG5349- 357 16 1 1 0.5 293-TG5349- 388 15 37 18 13.8 GP+E- 86 7 n.d. n.d.
GP + EnvAml2 n.d. 4 7 2.8 The clones 231, 275 and 280, which gave the highest titers in both experiments, were SUBST~TUTE SH'ET (RULE 26) W 0 97/35996 PCT~B96/00439 finally subcloned by limited dilution in 96 well plates using a cell suspension containing 0.3 cells per 200 ,ul per well. Subclones were retested for reverse transcliptase activity.
Those with highest RT activity were 231-6 and 275- 12.
Production of stable ~a~/pol and env cell lines Clones 293 TG5349 231-6 and 275- 12 were transfected in l O cm dishes with 'O lug pTG 5387 (4070A amphotropic env). After transfectioll al1d splitting of each dish into S new lO cm dishes, the cells were selected for XGPRT with 25 ~lg/ml mycophenolic acid. Medium was changed every 4 days and resistal1t colonies were picked into 24 well plates after 4 weeks. After the cultures had grown to confluency, the clones lO (approximately 250) were split into two wells of a six well plate. The cells from one well were subsequently trypsinized and frozen in DMEM 105roDMSO, 20%FCS, the cells inthe duplicate well were transfected with 2 ,ug of the retroviral vector pTG 5391 (LTR-LacZ-SV40prom-pac-LTR) ~nd tested for transient virus production 48 h after the tral1sfection by determining the ~-galactosidase titers on infected NIH 3T3 cells.
l 5 Additionally, after recovery of the medium, the transfected clones were selected for stable virus vector production in medium containing l~g/ml puromycin and the puromycin resistal1t pools were retitered.
Of the 250 clones screened by this procedure, approximately l in 5 clones produced a titer of greater than 105. Eight clones producing titers between 5x l o6 and Ix107 were finally chosen for subsequent cloning by limited dilution (parental clones A, B, C. D, E, F, G and H). Of these eight clones, two grew very well and generated fast glOWil1g subclones. 20 subclones for each parental clones A to H were tested for transient al1d stable virus vector production after transfection with pTG 5391 as before. The best 6 sLlbclones were additionally tested with another vector construct, pTG 9325 (LTR-Neo-25 LTR). The stable titers obtained from puromycin or neomycin-selected pools of cells are ~iven in table III.

SUBST~TU~E S~EET ~RULE 26) CA 02249987 l998-09-24 W O 97135996 PCT/Lbr''00139 TABLE III

pTG 5391 pTG 9325 s (lacZ) (Neomycin) PA317 0.9 X106 1.0 X106 293 TG5349 + TG5387 clone E2 1.8 X106 1.5 X106 clone E17 4.5 X106 3.5 X106 cloneE20 3.5 X106 0.3 X106 clone B4 2.0 X106 0.5 X106 clone B18 1.0 X106 0.4 X106 clone C26 1.0 X106 0.4 X106 TABLE IV

pTG 5391 (lacZ) PA317 subclone 4 0.5 x107 293 TG5349 + TG5387 subclone E2 -6 2.8 x10' subclone E2-12 4.0 x107 subclone E17-3 1.8 x10' subclone E17-5 1.7 x10' subclone E17-12 1.8 x10' subclone E20-2 2.3 x10' subclone E20-4 3.3 x107 subclone E20-7 1.8 x107 subclone E20-14 1.8 x107 SUBSTITUTE SHEET (RULE ~) WO 97/35996 pcTlIs96loo439 pTG5391 producer cells were subcloned by limited dilution. Individual subclones were isolated and the titers were compared to the llighest producer subclone that could be isolated from PA317 cells. The results (Table IV) show that individual prod-lcer subclones genelated from the 293 based gag/pol env cell line produce titers 5 wllich largely exceed 107 cfu/ml.
Transduction capabilities of vinls produced Oll the 293 the packatin~ cells.
To determine the transduction efficiency of vectors produced by a cell line of the presellt inventioll to currently used systems, we infected mouse NIH 3T3 cells, Mus dunlli tail fibroblasts Iwhich, unlike NIH 3T3 cells, are not infectable by ecotropic 10 vh-uses), h~lman lung carcinoma cells A549, human HeLa cells, canine MDCK cells and feline PG4 cells with supernatants of producer clones 293 E17-3 and PA317-4, and with the supernatant from a producer clone of GP+EnvAm 12 cells cotransfected with vector pTG 4371 and a plasmid encoding the puromycin resistance marker. Producer cell clones were seeded in 175 cm2 cell culture flasks. When cultures were 80% confluent, the 15 medium was removed, replaced by 8 ml of fresh medium, and 24 h later supernatants were recovered, and filtered through 0.45,um filters. The supernatants were supplemented with 15 llg/ml of polybrene and 0.5 ml was subsequently used to infect the different target cells which were 50-60~o confluency in 6 well plates. I h later, 3 ml of medium was added. and after 24 h the medium was changed. After 48 h the celis were stained for ~-20 galactosidase activity. As can be seen in Fig. 7, vectors from 293 packaging cellstr.lnsduce the different cells 5- 10 times more efficiently than vectors produced on PA317 or GP+EnvAml2 cells. This, in part may be due to the higher titers produced on 293 based packaging cells. However, as was described by Morgan, et al. (J. Virol. 69:6994-7000~ 1995) and Forestell et al. (Gelle T~lerapv 2:723-730, 1995), there is no direct linear '5 correlation between the multiplicity of infection and the transduction efficiency or between the end-point-titer determined on NIH 3T3 cells and the transduction efficiency of cells different from NIH 3T3 cells. Additional, specific factors from 293 cells may thu.~i add to the significant increase in transduction efficiency observed with the cells of the SUBSTITUTE SHEET (RULE 26) -Wo 97/35996 PcT/Isg6/00439 present invention.
Stability of the 293 packaging cell lines E 17-3 and PA317-4 Producer clones E 17-3 and PA317-4 were passaged every 4 days at a ratio of 1: 10 hl medium without any selective antibiotics. Every 5 passages, cells were frozen.
5 Subsequelltly, the cells with different passa~,e numbers were thawed in parLIllel. The supelnatants obtained from these cells were Litered on NIH 3T3 for ~-g~lactosidase activity. Titers of 293 producer were shown to be stable for at least 10 passages whereas titers from PA317 producer cells dropped by a factol- of 2.
Industrial Applications The procedures described above can be used to produce virion which display on theh- sulface envelope glycoproteins from any of a wide variety of retroviruses incl-lding polytropic, xenotropic, or ecotropic vims. For example, suitable retroviral envelope proteins include those derived from Feline Leul;emia virus, Gibbon ape Leukemia virus (Eglitis et al., Ge/le Tllerap.Y 2:486, 1995; Miller et al., J. Virol. 65:~220, 15 1991), or other retroviral leukemia virus. Gibbon ape leukemia virus envelope is a particularly desirable envelope protein because it recognizes many human cell types, in particular, bone marrow cells. Envelope proteins which mediate the infection or progenitor cells are particularly useful for somatic cell therapy.
ln general, envelope proteins from any enveloped virus may be used, whether 20 the virus is a DNA virus, a negative strand RNA virus or a positive strand RNA virus.
Thus, useful envelope proteins include, but are not limited to, those of: Vesicular Somatitus Virus (VSV) G protein, hepatitis B glycoprotein, influenza virus HA
glycoprotein.
The procedures described above can be used to create packaging cell lines for ~5 the production of pseudotyped virus in which the genome of a first virus is associated with the envelope protein of a second virus. Such virus can be used to infect cells recognized by the envelope protein of the second virus. In some circumstances it is preferable to use hybl-id envelope proteins produced by combining dolnains from two or morc different ~4 S~BSTITUTE Sl~~E~ (R~LE 26) genes such that the cytoplasmic domain of the envelope is derived from a virus that is compatible with the remainder of the viral components and the extracellular portion of the envelope is derived from a virus which can infect the target cells. The preferred location for recombillation is within the membrane spanning region of the envelope protein. Jolly 5 el al. (wo 92/05~66) describes methods for producillg hybl-id envelope proteins.
The methods described above can also be usecl to prepale virus having other genetically modified envelope proteins.
Cell lines other than humall 293 cells can be used to prep~re packaging cell lines. Preterred cell lines are those which are relatively or completely free of endogenous 10 retl-oviral sequences.
Under some circumstances it may be desirable to produce retrovirus by transient transfection of a packaging cell line rather than by the production of a stably transfected producer cell line. This approach is particularly useful for producing high tiers of retroviral vectors which express gene that are difficult to propagate at high tier in stable 15 producer cell lines. Pear et al. (Proc. Natl. Acad. Sci. USA 90:8392, 1993) describes the use of transient transfection to produce retrovirus.
Packaging cell lines can be used to generate producer cell lines which can be used to produce virions for the transfection of target cells. Target cells can be transduced by co-cultivation with producer cells or by incubation with viral particles or viral ~0 supernatant obtained by the culturing of producer cells. Target cells are generally exposed to virions in the presence of polycation (Cornetta et al., J. vi/ol Metlz. 23: 187, 1989).
Repeated transduction can be used to increase the percentage of target cell transduced.
The use of vectors which include selectable markers followed by positive selection can also increase the percentage of target cells transduced.
Rosenberg et al. (Ne~ gl. J. Med. 323, 1990) describes a useful gene therapy protocol employing a retroviral vector. In order to be useful for gene therapy, a plodLIcer cell line must not generate replication competent virus. A number of methods fol determining wheti1el a retroviral sllpernatanl is free of replication competent retrovilus ~5 SlJBSTITUTE SHEET (RULE 2~) W O 97135996 PCT~B96/00439 are known to those skilled in the art. In one approach producer cell supernatant is used to transdLIce a susceptible target cells which are then te~ted by PCR for the presence of helpel- virus DNA (Morgan et al., Hl(l)tall Gefle Tller. 1:135, 1990; Anderson et al., H~ lclll Gelle Tllerap~ 4:31 1, 1993). This approach is capable of detecting one cell 5 contail1ing helper vims against a background of 100,000 helper vims-free transduced cells. Like all other therapeutic agents, retrovital supernatant should be tested for the presence of potential pathogens and toxins using the standard FDA required general safety tests. Transduced cell are generally suspended in normal saline for administration to a patient by subcutaneous injection. Anderson et al. (U.S. 5,399,346) describes a number of 10 useful techniques for the preparation of transduced cells for administration to patients.

What is claimed is:

~UBSTlTU ~ E S~EET (RULE 26)

Claims (23)

1. A packaging cell line harboring a first expression plasmid encoding functional Friend Murine Leukemia Virus gag/pol.

2. The packaging cell line of claim 1 wherein said cell line harbors a second expression plasmid encoding a functional viral envelope protein.

3. The packaging cell line of claim 2 wherein said viral envelope protein is a retroviral envelope protein.

4. The packaging cell line of claim 2 wherein said viral envelope protein is a non-retroviral envelope protein.

5. The packaging cell line of claim 1 wherein the cells of said cell line are transiently transfected with said first expression plasmid.

6. The packaging cell line of claim 2 wherein the cells of said cell line are transiently transfected with said second expression plasmid.

7. The packaging cell line of claim 1 wherein the cells of said cell line are stably transfected with said first expression plasmid.

8. The packaging cell line of claim 2 wherein the cells of said cell line are stably transfected with said second expression plasmid.

9. The packaging cell line of claim 2 wherein the cells of said cell line are stably transfected with said first expression plasmid and said second expressionplasmid.

10. The packaging cell line of claim 9 wherein said first expression plasmid is integrated into the genome of the cells of said cell line at a first location and said second expression plasmid is inserted into the genome of the cells of said cell line a second location distinct from said first location.

11. The packaging cell line of claim 1 wherein said Friend Murine Leukemia Virus is strain FB29.

12. The packaging cell line of claim 2 wherein said envelope is an amphotropic envelope.

13. The packaging cell line of claim 2 wherein said envelope is an ecotropic envelope.

14. The packaging cell line of claim 2, said cell line being produced by transfecting human 293 cells with said first expression plasmid to obtain a gag/pol expressing cell line and then transfecting said gag/pol expressing cell line with said second expression plasmid.

15. The packaging cell line of claim 2, said cell line being produced by transfecting human 293 cells with said second expression plasmid to obtain an envelope expressing cell line and then transfecting said envelope expressing cell line with said first expression plasmid.

16. The packaging cell line of claim 15 wherein the cells of said cell line are stably transfected with said first and said second expression plasmid.

17. The packaging cell line of claim 16 wherein the cells of said cell line are stably transfected with said first and said second expression plasmid.

18. The packaging cell line of claim 2 wherein said first an second expression plasmids do not include a functional retroviral LTR.

19. The packaging cell line of claim 2 said cell line further harboring a retroviral vector, said cell line being capable of producing virion.

20. Viron produced by the cell line of claim 20.

21. A method for producing virion comprising:
(a) stably transfecting the packaging cell line of claim 2 with a selected replication incompetent retroviral vector which comprises a packaging sequence to create a producer cell line; and (b) culturing said producer cell line under conditions causing production of virion.

22. The method of claim 22 wherein said packaging sequence is not the Friend Murine Leukemia Virus FB29 psi sequence.

23. The method of claim 23 wherein said packaging sequence is a retrotransposon virus-like 30 S RNA psi sequence.