AU1699001A - Permanent amniocyte cell line, the production thereof and its use for producing gene transfer vectors - Google Patents

Abstract

Die Erfindung betrifft eine permanente Amniozyten-Zelllinie, enthaltend mindestens eine Nukleinsäure, die die Expression der Genprodukte der E1A- und E1B-Region von Adenovirus bewirkt. Weiterhin betrifft die vorliegende Erfindung die Herstellung einer permanenten Amniozyten-Zelllinie und ihre Verwendung zur Herstellung von Gentransfer-Vektoren. Weitere Aspekte sind die Verwendung von Amniozyten bzw. der adenoviralen Genprodukte der E1A- und E1B-Region zur Herstellung von permanenten Amniozyten-Zelllinien.

Description

WO 01/36615 - 1 - PCT/EPOO/10992 Permanent amniocytic cell line, its production and use for the production of gene transfer vectors 5 The present invention relates to a permanent amniocytic cell line comprising at least one nucleic acid which brings about expression of the gene products of the adenovirus ElA and ElB regions. The present 10 invention further relates to the production of a permanent amniocytic cell line and to its use for producing gene transfer vectors. Further aspects are the use of amniocytes and of the adenoviral gene products of the ElA and ElB regions for producing 15 permanent amniocytic cell lines. Adenoviruses Adenoviruses are a relatively homogeneous group of viruses characterized by an icosahedral capsid which 20 consists mainly of the virally encoded hexon, penton and fiber proteins, and of a linear, double-stranded DNA genome with a size of about 36 kilobases (kb) . The viral genome contains at the ends the inverted terminal repeat sequences (ITRs) which comprise the viral origin 25 of replication. There is furthermore at the left-hand end of the genome the packaging signal which is necessary for packaging of the viral genome into the virus capsids during an infection cycle. Adenoviruses have been isolated from many species. There are more 30 than 40 different human serotypes based on parameters which discriminate between the various serotypes, such as hemagglutination, tumorigenicity and DNA sequence homology (Wigand et al., in: Adenovirus DNA, Doerfler ed., Martinus Nijoff Publishing, Boston, pp. 408-441, 35 1986) . Adenoviral vectors to date are usually derived from serotypes 2 (Ad2) and 5 (Ad5) . Infections by Ad2 and Ad5 are endemic in humans. Ad2 and Ad5 are not oncogenic in humans and have good safety documentation because vaccinations have been performed on military - 2 personnel successfully and without complications in the USA (Pierce et al., Am. J. Epidemiol. 87, 237-246, 1968) . The biology of adenoviruses is relatively well understood because adenoviruses have played an 5 essential part in molecular biology as experimental tool for elucidating various fundamental biological principles such as DNA replication, transcription, RNA splicing and cellular transformation. Adenoviral particles enter the cell during an infection through 10 receptor-mediated endocytosis in which, according to the current view, interaction of the knob domain of the fiber protein with the coxsackie adenovirus receptor (CAR) mediates adhesion of the virus particle to the cell surface (Bergelson et al., Science 275, 1320-1323, 15 1997). In a second step there is internalization of the virus particle, for which interaction of the penton base with integrins plays an essential part (Wickham et al., Cell 73, 309-319, 1993). After the particle has entered the cell, the viral genome gets into the cell 20 nucleus as DNA-protein complex. The adenoviral infection cycle is divided into an early and a late phase which are separated by the start of adenoviral replication (Shenk, in: Virology, Fields ed., Lippincott-Raven Publishing, Philadelphia, pp. 25 2111-2148, 1996). In the early phase there is expression of the early viral functions El, E2, E3 and E4. The late phase is characterized by transcription of late genes which are responsible for the expression of viral structural proteins and for the production of new 30 viral particles. E1A is the first viral gene to be expressed by the viral chromosome after the cell nucleus is reached. The ElA gene codes for the 12S and 13S proteins which are formed by alternative splicing of the ElA RNA. The 35 ElA proteins activate the transcription of a number of cellular and viral genes by interacting with transcription factors. The main functions of ElA are a) activation of the other early viral functions ElB, - 3 E2, E3 and E4 and b) inducing resting cells to enter the S phase of the cell cycle. Expression of ElA on its own leads to programmed cell death (apoptosis). ElB is one of the early viral genes activated 5 by ElA. The ElB gene codes for the ElB 55 kD protein and the ElB 19 kD protein, which result through alternative splicing of the ElB RNA. The 55 kD protein modulates the progression of the cell cycle by interacting with the p53 tumor suppressor gene, is 10 involved in preventing the transport of cellular mRNA in the late phase of the infection, and prevents E1A induced apoptosis of cells. The ElB 19 kD protein is likewise important for preventing ElA-induced apoptosis of cells. 15 All human adenoviruses are able to transform rodent cells in cell culture. As a rule, coexpression of ElA and E1B is necessry for oncogenic transformation. The protein IX gene which codes for a 20 structural component of the viral capsid is embedded in the ElB transcription unit. The E2A and E2B genes code for various proteins which are essential for replication of the viral genome. These comprise the precursor protein of the 25 terminal protein (pTP), the DNA polymerase (Pol) and the single strand-binding protein (SSBP). On replication, pTP binds to the ITRs of the viral genome. There it acts as protein primer for DNA replication, which is initiated by Pol together with cellular 30 factors. Pol, SSBP and the cellular factor NFII, and presumably other factors, are necessary for DNA chain extension. E4 codes for various proteins. Inter alia, the E4 34 kD protein blocks, together with the ElB 55 kD 35 protein, the accumulation of cellular mRNAs in the cytoplasm, and at the same time it facilitates the transport of viral RNAs from the cell nucleus into the cytoplasm.

- 4 After the start of replication of the viral genome there is expression of viral structural proteins which are necessary for establishment of the viral capsid and for complexation of the viral DNA with 5 virally encoded DNA-binding proteins. There is evidently initial formation of an empty capsid, into which the viral genome subsequently enters. A cis element on the viral genome is necessary for this process, the so-called packaging signal which is 10 located at the left-hand end of the viral genome and, in the case of Ad5, extends over a region from base pair 260 to base pair 460 (Hearing et al., J. Virol. 62, 2555-2558, 1987; Graeble and Hearing, J. Virol. 64, 2047-2056, 1990) . The packaging signal overlaps with 15 the ElA enhancer which is essential for activity of the ElA promoter. The exact mechanism of the packaging of the viral genome into the virus capsids is not clear but it is probable that interaction of cellular and/or viral proteins with the packaging signal is necessary 20 for this. Adenovirus vectors Adenoviral vectors are particularly important as expression vectors, especially for the purpose of 25 gene therapy. There are several reasons for this: the biology of adenoviruses has been thoroughly investigated. The virus particles are stable and can be produced relatively simply and in high titers. Genetic manipulation of the adenoviral genome is easy. 30 Adenovirus vectors are able efficiently to transduce replicating and nonreplicating cells in vitro and in vivo. a) First-generation adenoviral vectors 35 First-generation adenoviral vectors (Gilardi et al., FEBS Letters 267, 60-62, 1990; Stratford Perricaudet et al., Hum. Gene Ther. 1, 241-256, 1990) are characterized by deletions of the ElA and ElB - 5 genes. ElA and ElB have transforming and transactivating properties. In addition, ElA is necessary for activating viral genes and ElB is necessary for the accumulation of viral transcripts. In 5 some vectors in addition E3 is deleted in order to increase the capacity for uptake of foreign DNA. E3 is dispensable for producing adenoviruses in cell culture. The capacity for uptake of foreign DNA is about 8 kb. First-generation adenovirus vectors have to date been 10 produced mainly in 293 cells (see below) which complement the ElA and ElB deficit of the vectors. b) Second-generation adenoviral vectors Second-generation adenoviral vectors are 15 characterized by deletions of E2 and/or E4 in addition to deletions of ElA and ElB (Engelhardt et al., Proc. Natl. Acad. Sci., USA 91, 6196-6200, 1994; Yang et al., Nature Genet., 7, 362-367, 1994; Gorziglia et al., J. Virol. 70, 4173-4178, 1996; Krougliak and Graham, 20 Hum. Gene Ther. 6, 1575-1586, 1995; Zhou et al., J. Virol. 70, 7030-7038, 1996). In some vectors in addition E3 is deleted in order to increase the capacity for uptake of foreign DNA. Second-generation adenoviral vectors were developed in order to reduce 25 further the transcription of viral genes and the expression of viral proteins and in order thus to diminish further the antiviral immune response. The capacity for uptake of foreign DNA is negligibly increased by comparison with first-generation 30 adenoviral vectors. Second-generation adenovirus vectors are produced in cell lines which, in addition to ElA and ElB, complement the particular deficit (E2 and/or E4).

- 6 c) Adenoviral vectors of large DNA capacity Adenoviral vectors of large DNA capacity are characterized by containing no viral coding DNA sequences (Kochanek et al., Proc. Natl. Acad. Sci. 5 U.S.A. 93, 5731-5736, 1996; Fisher et al., Virology 217, 11-22, 1996; Kumar-Singh and Chamberlain, Hum. Mol. Genet. 5, 913-921, 1996) . These vectors only contain the viral ends with inclusion of the ITRs and of the packaging signal. The capacity for uptake of 10 foreign DNA is about 37 kb because by far the major part of the adenoviral genome is deleted. Various systems have been described for producing adenoviral vectors of large DNA capacity (Kochanek et al., supra; Parks et al., Proc. Natl. Acad. Sci. USA 93, 15 13565-13570, 1996; Hardy et al., J. Virol. 71, 1842-1849, 1997). The advantage of these adenoviral vectors with large DNA capacity compared with first and second-generation adenoviral vectors is the larger capacity for uptake of foreign DNA and a lower toxicity 20 and immunogenicity (Schiedner et al., Nature Genet. 18, 180-183, 1998; Morral et al., Hum. Gene Ther. 9, 2709-2716, 1998). Currently, adenoviral vectors of large capacity are produced with the aid of an E1A- and ElB-deleted helper virus which provides the viral 25 functions necessary for a productive infection cycle in trans. To date, adenoviral vectors of large DNA capacity have been produced in 293 cells or in cell lines derived from 293 cells. In one of the production methods (Parks et al., supra; Hardy et al., supra), 30 adenoviral vectors are produced in modified 293 cells which, in addition to ElA and ElB, express the Cre recombinase of bacteriophage P1. In this system, the packaging signal of the helper virus is flanked by loxP recognition sequences of bacteriophage Pl. On infection 35 of Cre-expressing 293 cells with helper virus and the adenoviral vector of large DNA capacity, the packaging signal of the helper virus is excised. For this reason - 7 there is packaging mainly of the vector containing a normal packaging signal but not of the helper virus. d) Deleted adenoviral vectors 5 These vectors have been described as first generation vectors which have the loxP recognition sequences of bacteriophage P1 positioned in the viral genome in such a way that, on infection of Cre expressing 293 cells, most of the viral coding 10 sequences or all the viral coding sequences are deleted by recombination between the loxP recognition sequences. The size of the genome of these vectors is about 9 kb. The capacity for uptake of foreign DNA is likewise about 9 kb (Lieber et al., J. Virol. 70, 15 8944-8960, 1996). Adeno-associated virus (AAV) AAV belongs to the family of parvoviruses, genus dependovirus, and has two different life forms, 20 occurring either as lytic virus or as provirus. For a lytic infection to take place the virus requires coinfection with a helper virus (adenovirus, vacciniavirus, herpes simplex virus) . In the absence of a helper virus, AAV is unable to replicate, integrates 25 into the genome and exists there as inactive provirus. When cells harboring AAV as integrated provirus are infected, for example with adenovirus, the provirus is able to enter a lytic infection cycle again (Samulski, Curr. Opin. Genet. Dev. 3, 74-80, 1993). 30 AAV capsids contain a single-stranded, linear DNA genome with either positive or negative polarity. Several AAV serotypes exist. The serotype which has been investigated most is AAV-2. The genome of AAV-2 consists of 4680 nucleotides. The genome contains at 35 the ends inverted terminal repeat sequences (ITRs) having a length of 145 base pairs. The first 125 base pairs form a T-shaped hairpin structure consisting of two internal palindromes.

-8 The AAV genome codes for nonstructural replication (Rep) proteins and for structural capsid (Cap) proteins. The various replication proteins (Rep78, Rep68, Rep52, Rep40) are generated by using 5 different promoters (p5 and p19) and by alternative splicing. The various capsid proteins (VP1, VP2, VP3) are generated by alternative splicing using the p40 promoter. 10 AAV vectors AAV vectors contain only the ITRs of AAV and some adjacent, noncoding AAV sequences. For this reason, the capacity for uptake of foreign DNA is about 4.5 kb. Various systems have been described for 15 producing recombinant AAV vectors (Skulimowski and Samulski, in: Methods in Molecular Genetics, Vol. 7, Adoph ed., Academic Press, pp. 3-12) . The components necessary for replication, expression and packaging of the recombinant vector are provided in these systems. 20 Specifically, these are expression cassettes which code for the Rep and Cap proteins of AAV, and the adenoviral helper functions. The adenoviral helper functions necessary for AAV production are, specifically, ElA, ElB, E2, E4 and VA. The ElA and ElB functions are 25 provided in the 293 cells which have been used for production to date. In the production processes described to date, the E2, E4 and VA functions are currently usually provided either by coinfection with adenovirus or by cotransfection with E2-, E4- and VA 30 expressing plasmids (Samulski et al., J. Virol. 63, 3822-3828, 1989; Allen et al., J. Virol. 71, 6816-6822, 1997; Tamayose et al., Hum. Gene Ther. 7, 507-513, 1996; Flotte et al., Gene Ther. 2, 29-37, 1995; Conway et al., J. Virol. 71, 8780-8789, 1997; Chiorini et al., 35 Hum. Gene Ther. 6, 1531-1541, 1995; Ferrari et al., J. Virol. 70, 3227-3234, 1996; Salvetti et al., Hum. Gene Ther. 9, 695-706, 1998; Xiao et al., J. Virol. 72, 2224-2232, 1998, Grimm et al., Hum. Gene Ther. 9, - 9 2745-2760, 1998; Zhang et al., Hum. Gene Ther. 10, 2527-2537, 1999) . Alternatively, strategies have been developed in which adenovirus/AAV or herpes simplex virus/AAV hybrid vectors have been used to produce AAV 5 vectors (Conway et al., supra; Johnston et al., Hum. Gene Ther. 8, 359-370, 1997, Thrasher et al., Gene Ther. 2, 481-485, 1995; Fisher et al., Hum. Gene Ther. 7, 2079-2087, 1996; Johnston et al., Hum. Gene Ther. 8, 359-370, 1997) . It is common to all these processes 10 that ElA- and ElB-expressing 293 cells are currently used for production. Producer cell lines For safety reasons, adenoviral vectors intended 15 for use in humans usually have deletions of the ElA and ElB genes. Production takes place in complementing cell lines which provide the El functions in trans. Most adenoviral vectors to date have been produced in the 293 cell line. In recent years, further cell lines 20 which can be used to produce El-deleted adenoviral vectors have been produced. a) HEK 293 cells HEK 293 cells were for a long time the only 25 cells which could be used to produce El-deleted adenoviral vectors. HEK 293 cells were produced in 1977 by transfection of sheared adenoviral DNA into human embryonic kidney cells (HEK cells). In a total of eight transfection experiments each with an average of 20 HEK 30 cultures it was possible to obtain only a single immortalized cell clone (Graham et al., J. Gen. Virol. 36, 59-74, 1977). The cell line (HEK 293 cells) established from this cell clone contain [sic] the complete left-hand 11% of the adenoviral genome (base 35 pair 1 to 4344 of the Ad5 genome), including the ElA and ElB genes and the left-hand ITR and the adenoviral packaging signal (Louis et al., virology 233, 423-429, 1997). A considerable problem for the production of - 10 adenoviral vectors is the sequence homology between El deleted adenoviral vectors and the portion of adenoviral DNA integrated into 293 cells. Homologous recombination between the vector genome and the 5 adenoviral DNA integrated into 293 cells is responsible for the generation of replication-competent adenoviruses (RCA) (Lochmller et al., Hum. Gene Ther. 5, 1485-1491, 1994; Hehir et al., J. Virol. 70, 8459-8467, 1996) . HEK 293 cells are for this reason 10 unsuitable for producing adenoviral vectors of pharmaceutical quality because production units are often contaminated with unacceptable amounts of RCA. RCA is unacceptable in products produced for clinical use because replication-competent adenoviruses have a 15 distinctly higher toxicity than replication-defective adenoviruses, are capable of uncontrolled replication in human tissues, and are moreover able to complement replication-defective adenoviruses (Lochmller et al., supra; Imler et al., Hum. Gene Ther. 6, 711-721, 1995; 20 Hehir et al., supra). b) Human embryonic retinal cells (HER cells) and established cell lines Although rodent cells can easily be transformed 25 with adenoviral El functions, primary human cells have proved to be relatively resistant to transformation with ElA and ElB. As mentioned above, Graham and coworkers were able to isolate only a single cell clone from HEK cells which had been transfected with sheared 30 Ad5 DNA. Gallimore and coworkers attempted for a long time unsucessfully to transform primary HEK cells with El functions of Ad12 (Gallimore et al., Anticancer Res., 6, 499-508, 1986). These experiments were carried out unsuccessfully over a period of three years with 35 more than 1 mg of the EcoRI-C DNA fragment of Ad12 containing the ElA and ElB genes. After many attempts it was possible, despite a large number of experiments carried out, to isolate only four Ad12-El HEK cell - 11 lines (Whittaker et al., Mol. Cell. Biol., 4, 110-116, 1984). Likewise, Gallimore and coworkers attempted unsuccessfully to transform other primary human cells with El functions, including keratinocytes, skin 5 fibroblasts, hepatocytes and urothelial cells (Gallimore et al., Anticancer Res., 6, 499-508, 1986). The only human cell type which it has been possible to date to transform reproducibly with adenoviral El functions comprises human embryonic retinal cells (HER 10 cells) . HER cells are a mixture of cells derived from the white neural retina. To obtain these cells it is necessary to remove the eye from the orbital cavity of a human fetus, normally between weeks 16 and 20 of gestation. The eye is opened with a horizontal incision 15 and the white neural retina can be removed with forceps and placed in cell culture. Based on earlier observations that a) Ad12 induced tumors are primarily derived from primitive neural epithelium (Mukai et al., Prog. Neuropathol. 3, 20 89-128, 1976) and that b) Ad12 induces retinal tumors in rats and baboons after intraocular inoculation (Mukai et al., supra; Mukai et al., Science 210, 1023-1025, 1980), Byrd and coworkers found that human embryonic retinoblasts (HER cells) can be transformed 25 with the El genes of Ad12 (Byrd et al., Nature 298, 69-71, 1982). Although the efficiency of transformation of HER cells was less than that of primary rat cells, the efficiency of transformation was more than 100 times higher than that of HEK cells. The investigations 30 were initiated in order to produce complementing cell lines which could be used to isolated Ad12 El mutants. In further investigations by this research group (Gallimore et al., Cancer Cells 4, 339-348, 1986) it was shown that HER cells can be transformed 35 efficiently with plasmid DNA which expresses the ElA and ElB genes of Ad5. The efficiency of transformation and the establishment of E1A- and ElB-expressing cell - 12 lines was about 20 times higher with the El genes of Ad5 than with El genes of Ad12. Based on these data, Fallaux and coworkers (Fallaux et al., Hum. Gene Ther. 7, 215-222, 1996; 5 Fallaux et al., Hum. Gene Ther. 9, 1909-1917, 1998) established ElA- and ElB-expressing cell lines by transforming HER. cells with plasmids which expressed the ElA and ElB genes of Ad5. The cell line 911 was produced by transformation with a plasmid which 10 contains the ElA and ElB genes of Ad5 (nucleotides 79-5789 of the Ad5 genome) and expresses ElA under the control of the natural ElA promoter (Fallaux et al., supra; Patent Application W097/00326) . It was possible to establish further E1A- and ElB-expressing HER cell 15 lines by transfecting a plasmid which contains nucleotides 459-3510 of the Ad5 genome, in which the ElA gene is under the control of the human phosphoglycerate kinase (PGK) promoter, and in which the natural ElB polyadenylation signal is replaced by 20 the poly(A) sequence of the heptatitis B virus (HBV) surface antigen (Fallaux et al., supra; Patent Application W097/00326). These HER cell lines have been referred to as PER cell lines. The advantage of these newer PER cell lines compared with 293 cells or the 911 25 cell line is the lack of sequence homology between the DNA of first-generation adenoviral vectors and the integrated Ad5 DNA. For this reason there is a marked reduction in the possibility of the generation of RCA. These ElA- and ElB-transformed HER cell lines (911 30 cells and PER cells) were able to complement the El deficit of first-generation adenoviral vectors and thus be used to produce these vectors. In a similar way, a cell line which was established by transforming HER cells with the plasmid 35 pTG6559 is mentioned in a publication by Imler and coworkers (Imler et al., supra; see also WO 94/28152) . The plasmid pTG6559 contains the coding sequences of the ElA and ElB genes and of the protein IX gene - 13 (nucleotides 505-4034 of the Ad5 genome) , with the ElA gene being under the control of the mouse phosphoglycerate kinase (PGK) promoter, and the joint polyadenylation signal of the ElB and protein IX genes 5 having been replaced by the polyadenylation signal of the rabbit -globin gene. In contrast to the described attempts to establish primary human cells by transformation with the ElA and ElB genes of Ad5, attempts have been made 10 in a few cases to express ElA and ElB of various serotypes stably in previously established cell lines (Grodzicker et al., Cell, 21, 453-463, 1980; Babiss et al., J. Virol. 46, 454-465, 1983; Shiroki et al., J. Virol. 45, 1074-1082, 1983; Imler et al., supra; see 15 also WO 94/28152). The disadvantages of these cell lines is [sic] the need for coexpression of a selection marker and the frequently deficient stability of ElA and ElB expression. Since these cell lines are immortalized cell lines from the outset, expression of 20 ElA and ElB is not necessary for survival of the cell lines, so that natural selection by ElA and ElB is unnecessary in this case and in contrast to the use of primary cells. In the past, the production of cell lines for 25 producing adenoviral vectors or for producing AAV vectors was associated with particular difficulties. Human embryonic kidney cells (HEK cells) can be obtained from the kidney of human fetuses. This is done by removing a kidney from a fetus and placing kidney 30 cells in the cell culture. Transfection of HEK cells with sheared Ad5 DNA and integration of the left-hand end of the Ad5 DNA, and expression of the ElA and ElB genes resulted in transformation of the cells in a single published case. It was possible to establish a 35 single cell line (293 cells) in this way (Graham et al., supra; see above "Producer cell lines", section a) . 293 cells are used to produce adenoviral vectors and to produce AAV vectors.

- 14 Human embryonic retinal cells (HER cells) can be obtained from the eyeball of human fetuses. This is done by removing an eye from the fetus and placing cells from the retina in culture. It was possible by 5 transfecting HER cells with the adenoviral ElA and ElB genes to transform HER cells (see above "Producer cell lines", section b) . Cells transformed with ElA and E1B can be used to produce adenoviral vectors. It is necessary in both cases to remove an 10 organ from human fetuses, which are derived either from a spontaneous or therapeutic abortion or from a termination of pregnancy on social .grounds, and to establish a cell culture from this organ. After establishment of a primary culture, these cells can 15 then be transformed by transfection with the adenoviral ElA and ElB genes. Cell lines established in this way and expressing ElA and ElB can then be used to produce adenoviral vectors or AAV vectors. It is evident that it is complicated to obtain 20 primary cells from organs from fetuses. Since a primary culture can be established only from fresh tissue, special logistic efforts are needed to obtain suitable tissue. In addition, the use of fetal tissue derived either from a spontaneous abortion, a therapeutic 25 abortion or from a termination of pregnancy on social grounds makes special ethical considerations and care necessary for establishment of a primary culture. Although the inventors' laboratory is situated in a gynecology clinic where terminations of pregnancy are 30 frequently performed, it was not possible to obtain suitable tissue over a period of more than one year. Removal of fetal tissue after abortion requires a declaration of consent by the pregnant woman after receiving appropriate information. It was frequently 35 impossible to obtain the consent of the pregnant woman for the organ-removal intervention after she had received detailed information about the project, i.e.

- 15 the removal of an eye from the fetus for scientific medical investigations. The use of a permanent amniocytic cell line for producing gene transfer vectors has not previously been 5 described. There have merely been a report of human amniocytes which have been transformed with the simian virus (SV40) and/or the Kirsten sarcoma virus (Sack, In Vitro 17 pp. 1-19, 1981; Walen, et al., In Vitro Cell Dev. Biol. 22, 57-65, 1986) . Infection with SV40 alone 10 conferred an extended lifetime (called immortalization) , whereas infection with the Kirsten sarcoma virus alone did not extend the lifespan. Infection with both viruses finally led to a malignant tumor cell (Walen and Arnstein, supra) . It should be 15 noted in this connection that SV40-transformed amniocytic cell lines are unsuitable for producing gene transfer vectors because these cells themselves produce SV40, which is known to be an oncogenic virus (Graffney et al., Cancer Res. 30, 871-879, 1970). The 20 transformability of human cells with SV40 moreover provides no information about the transformability with the El functions of adenovirus and the use thereof for the production of gene transfer vectors. For example, keratinocytes can be transformed with SV40 (see Sack, 25 supra) , but keratinocytes evidently cannot, just like skin fibroblasts and hepatocytes, be transformed with Ad12 (Gallimore et al., 1986, supra) . In terms of the production of viral vectors, especially adenoviral vectors, in immortalized cells, moreover, it is not 30 just the immortalizability with the particular immortalization functions which is important; so too are good infectability and a good productive course of infection. These properties cannot be predicted; the question of whether a particular cell type can be used 35 for producing gene transfer vectors must be determined anew for each cell type. An object of the present invention was therefore to provide a novel process for the efficient, - 16 simple and easily reproducible production of an amniocytic cell line, and the use thereof inter alia for producing adenoviral vectors, AAV vectors and retroviral or lentiviral vectors. 5 It has been found, entirely surprisingly, that transfection of cells of the amniotic fluid (amniocytes), which are routinely obtained by amniotic fluid biopsy (amniocentesis) for diagnostic reasons during prenatal diagnosis, with the adenoviral ElA and 10 ElB genes led to a large number of permanent cell lines which expressed the ElA and ElB genes in a functionally active manner and which are suitable for producing gene transfer vectors. One aspect of the present invention is 15 therefore a permanent amniocytic cell line comprising at least one nucleic acid which brings about expression of the gene products of the adenovirus ElA and ElB regions. A "permanent cell line" means according to the present invention that the corresponding cells have 20 been genetically modified in some way so that they are able to continue growing permanently in cell culture. By contrast, "primary cells" mean cells which have been obtained by removal from an organism and subculturing and which have only a limited lifetime. A permanent 25 amniocytic cell line for the purpose of the present invention can be obtained by the process proposed herein, which comprises the transfection of primary amniocytes with the El functions of adenovirus. The at least one nucleic acid which brings about expression of 30 the adenovirus El gene products can be any suitable nucleic acid or nucleic acids which lead to stable expression of these gene products. It/they can be integrated into the genome of the cell, i.e. chromosomally, or be present outside the chromosome, 35 for example as episomally replicating plasmid or minichromosome. Expression of the various gene products can moreover be brought about by one and the same nucleic acid molecule or, for example, different - 17 nucleic acid molecules. "Expression" means in the state of the art the process of production of a gene product which is either [sic] a specific protein which brings about a specific trait or a specific property, or of 5 RNA forms which are not translated into proteins (for example antisense RNAs, tRNAs) . Suitable possibilities for achieving the desired expression will be evident to the skilled worker in the light of the present description, in particular of the proposed process too. 10 The novel amniocytic cell line is suitable not only for use for producing gene transfer vectors in general but also, in particular, for producing first-generation adenoviral vectors characterized by deletions of the ElA and ElB genes, which are complemented by the cell 15 line. The at least one nucleic acid also preferably brings about expression of the gene products of the adenovirus E2A, E2B and/or E4 regions and/or of Cre recombinase. This makes the cell line particularly 20 suitable for producing second-generation adenoviral vectors which are characterized by deletions of E2 and/or E4 genes in addition to the deletions of the ElA and ElB genes. Expression of the Cre recombinase of bacteriophage P1 is particularly advantageous in the 25 production of adenoviral vectors of large capacity with the aid of an ElA- and ElB-deleted helper virus (see also Parks et al., supra; Hardy et al., supra). Expression of the gene products of the E1A region is advantageously under the control of a constitutive 30 promoter, preferably the phosphoglycerate kinase (PGK) promoter. It is advantageous for expression of the gene products of the ElB region if it is under the control of an adenoviral promoter, preferably the adenoviral ElB promoter. A possible alternative to this is to 35 employ, for example, a cytomegalovirus (CMV) promoter. All the adenoviral gene products are preferably derived from an adenovirus of the same subgenus, for example of human adenovirus type 5 (Ad5) . The permanent amniocytic - 18 cell line is normally a human cell line, because this is particularly suitable for producing gene transfer vectors derived from human viruses, such as, for example, a human adenovirus or a human AAV. 5 A possible alternative to this is a cell line from primates or other mammals such as, for example, bovines, which are [sic] particularly suitable for producing gene transfer vectors derived from viruses occurring and endemic in particular species. For 10 example, permanent amniocytic cell lines obtained by transformation of amniocytes with the ElA and ElB genes of a bovine adenovirus are suitable for producing vectors derived from a bovine adenovirus. Another aspect of the present invention is a 15 process for producing a permanent amniocytic cell line, in particular an amniocytic cell line as defined above, which comprises the transfection of amniocytes with at least one nucleic acid which brings about expression of the adenoviral gene products of the ElA region and ElB 20 region. The resulting cell clones can then be isolated further where appropriate and, if required, be cloned to obtain single cell lines. The term "transfection" means herein any process suitable for introducing said nucleic acid(s) into the cells. Examples which may be 25 mentioned are electroporation, liposomal systems of any type and combinations of these processes. The term "amniocytes" means herein in the wider sense all cells which are present in the amniotic fluid and can be obtained by amniotic fluid biopsy. They are derived 30 either from the amnion or from fetal tissue which is in contact with the amniotic fluid. Three main classes of amniocytes have been described and are distinguished on the basis of morphological criteria: fibroblast-like cells (F cells), epitheloid cells (E cells) and 35 amniotic fluid cells (AF cells) (Hohn et al., Pediat. Res. 8, 746-754, 1974). AF cells are the predominant cell type. In the narrow sense, therefore, "amniocytes" mean herein amniocytes of the AF type. Primary - 19 amniocytes are preferably used. Cells referred to as "primary" cells are those which can be obtained by removal from an organism and subculturing and have only a limited lifetime, whereas so-called "permanent" cell 5 lines are able to continue to grow unrestrictedly. It is particularly preferred in this connection to use human primary amniocytes which lead to the production of human cell lines (see above). However, it is also possible to use primary amniocytes from primates and 10 other mammalian species such as from bovines. It will also be evident to the skilled worker in the light of the present description that it is possible to use analogously cells which can be obtained from the amniotic membranes, for example by trypsinization, or 15 by a chorionic villus biopsy, for producing corresponding permanent cell lines. The at least one nucleic acid which brings about expression of the adenoviral El gene products can be genomic DNA, cDNA, synthetic DNA, RNA and mRNA. The 20 nucleic acid is preferably used in the form of a DNA expression vector. Examples thereof are integrative vectors, bacterial plasmids, episomally replicated plasmids or minichromosomes. Preference is given to expression plasmids whose integration into the genome 25 of the recipient cell is brought about by transfection. The term "at least one nucleic acid" expresses the fact that the elements which bring about the expression may be present either on one and the same nucleic acid or on different nucleic acids. For example, separate 30 nucleic acids may be provided for expression of the gene products of the ElA, ElB, E2A, E2B and/or E4 regions and/or of Cre recombinase. It is also conceivable that the amniocytes to be transfected already express one of these gene products so that only 35 the expression of the other gene product(s) needs to be brought about, or that the expression of one or more of these gene products is switched on merely by introducing suitable regulatory elements. Suitable - 20 techniques and processes for the production and, where appropriate, mutagenesis of nucleic acids and for gene expression and protein analysis are available to the skilled worker (see, for example, Sambrook, J. et al., 5 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989); Glover, D.M., DNA cloning: A practical approach, vol. II: Expression Systems, IRL Press (1995); Ausubel et al., Short protocols in molecular biology, John Wiley & Sons 10 (1999); Rees, A.R. et al., Protein engineering: A practical approach, IRL press (1993)). It is preferred for the gene product or gene products of the ElA region to be expressed under the control of a constitutive promoter, in particular the phosphoglycerate kinase 15 (PGK) promoter, and for the gene products of the ElB region to be expressed under the control of an adenoviral promoter, in particular the adenoviral ElB promoter. In place of the adenoviral promoter it is also possible to use, for example, a cytomegalovirus 20 (CMV) promoter. In a particular embodiment, transfection of the amniocytes and/or of the resulting cell line additionally brings about expression of the resulting gene products [lacuna] adenovirus E2A and/or E2B and/or 25 E4 regions and/or of Cre recombinase. All the possibilities discussed previously or otherwise disclosed in the prior art are available to the skilled worker in this connection. Concerning the individual genes, reference is made in addition to the following 30 information: E2A: Genbank Acc. #M73260; Kruiyer et al., Nucl. Acids Res. 9, 4439-4457, 1981; Kruiyer et al., Nucl. Acids Res. 10, 4493-4500, 1982. E2B: Genbank Acc. #M73260; Dekker et al., Gene 27, 115-120, 1984; Shu et al., Virology 165, 348-356, 1988. E3: Genbank Acc. 35 #M73620; Cladaras et al., Virology 140, 28-43, 1985. E4: Genbank Acc. #M73620 and D12587; Virtanen et al., J. Virol. 51, 822-831, 1984; Dix et al., J. Gen. Virol. 73, 2975-2976, 1992. The reading frames are in some - 21 cases known only for Ad2 and can then usually be assigned by comparison of sequences in the case of, for example, Ad5. Cre recombinase: Genbank Acc. #X03453; Sternberg et al., J. Mol. Biol. 187, 197-212, 1986. 5 The adenoviral gene products are preferably all derived from a particular adenoviral serotype, in particular from human adenovirus type 5 (Ad5). The particular adenoviral serotype which is the origin of the ElA and ElB genes used for transforming amniocytes 10 is not critical for this invention. Example of adenoviral serotypes which can be used in the present invention are known in the prior art and include more than 40 human serotypes, for example Ad12 (subgenus A), Ad3 and Ad7 (subgenus B), Ad2 and Ad5 (subgenus C), Ad8 15 (subgenus D), Ad4 (subgenus E), Ad40 (subgenus F) (Wigand et al., in: Adenovirus DNA, Doerfler, ed., Martinus Nijhoff Publishing, Boston, pp. 408-441, 1986). In a preferred embodiment of this invention, adenoviral vectors derived from subgenus C are produced 20 by transforming amniocytes with ElA and ElB genes which are derived from an adenovirus of the same subgenus. For example, adenoviral vectors of serotype 2 or 5 are produced by transforming amniocytes with the ElA and E1B genes of serotype 2 or 5. Adenoviral 25 vectors based on Ad12 are produced by transforming amniocytes with the ElA and ElB genes of Ad12 etc. To produce non-human adenoviral vectors, including of the well-known adenoviruses derived from cattle, sheep, pigs and other mammals, for the production [sic] 30 amniocytic cell lines are produced by transforming amniocytes of the particular species. This is usually necessary because adenoviral functions usually cannot be complemented efficiently beyond species boundaries. In a particular embodiment of the invention, 35 amniocytes obtained for diagnostic reasons within the scope of prenatal diagnosis by amniotic fluid biopsy and no longer used for diagnostic purposes were transfected with an expression plasmid which expressed - 22 the ElA and ElB genes of Ad5. This construct was designed so that the ElA gene was under the control of the mouse phosphoglycerate kinase promoter, and the ElB gene was under the control of the natural adenoviral 5 ElB promoter. The natural ElB splice acceptor site and the ElB polyadenylation sequence were replaced by the corresponding sequences of the SV40 virus. A few weeks after transfection with the plasmid DNA, a large number of cell clones was observed, and these were isolated, 10 cloned, established as immortalized cell lines and analyzed. All the analyzed cell clones expressed the ElA and ElB proteins. It was shown, by infection with an El-deleted, -gal-expressing adenoviral vector and subsequent staining, that all these cells could be 15 infected. Infection experiments with El-deleted first generation adenoviral vectors revealed that the cell lines are suitable for producing adenoviral vectors. In these experiments, the cell lines were initially infected with a P-gal-expressing first-generation 20 adenoviral vector. After 48-72 hours, when the cells showed a cytopathic effect (CPE), the cells were harvested and the adenoviral vector was freed of cells by freezing and thawing three times. Part of the cell lysate was used to infect 293 cells, and -gal 25 expression was detected histochemically about 24 hours after the gene transfer. It was possible to calculate directly from the number of -gal-positive cells the yield of the vector by production in the individual cell lines. Amniocytic cell lines can be obtained in 30 this way without difficulty and reproducibly and are very suitable for producing gene transfer vectors. Some of the isolated cell lines allowed adenoviral vectors to be produced just as well as or better than 293 cells. As was to be expected, the cell lines showed 35 differences in the production of recombinant adenovirus vector (see Fig. 4). The cell lines N52.E6 and N52.F4 were distinguished by a rapid growth and particularly good production of adenoviral vectors, beneficial - 23 properties for the use of these cell lines for producing gene transfer vectors. The design of the ElA- and ElB-expressing expression plasmid used for transforming the amniocytes 5 precludes the generation of replication-competent adenoviruses (RCA) by homologous recombination of an adenoviral vector or of an adenoviral helper virus with the DNA integrated into the transformed amniocytes, in contrast to 293 cells. As an alternative to this, the 10 individual El functions can be introduced on various expression plasmids into the cells to be transfected. It is, of course, also possible, as for the 293 cell line, to carry out a transformation of amniocytes and to test the batches generated in the production of gene 15 transfer vectors for the RCA content, for example using a PCR or infection assay. The RCA-containing batches can then be discarded where appropriate. Thus a further aspect of the present invention relates to a permanent amniocytic cell line which can 20 be obtained by the process proposed herein. In a specific embodiment, the invention relates to the permanent amniocytic cell line N52.E6 which was deposited on Oct. 26, 1999 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) in 25 accordance with the Budapest treaty and has received the accession number DSM ACC2416. In a further aspect, the present invention relates to the use of amniocytes for producing adenovirus-transformed permanent amniocytic cell lines. 30 The term "adenovirus-transformed" means herein transformation by one or more transforming adenovirus genes. "Transformation" refers in this connection to conversion of a eukaryotic cell which is subject to growth control into a so-called permanent cell line 35 which grows unrestrictedly. A further aspect is the use of the adenoviral gene products of the ElA and ElB regions for producing permanent amniocytic cell lines.

- 24 The present invention further comprises the use of a permanent amniocytic cell line for producing gene transfer vectors. "Gene transfer vectors" mean herein generally all vectors with which one or more 5 therapeutic genes can be transferred or introduced into the desired target cells and, in particular, viral vectors having this property. In addition, the permanent amniocytic cell lines can be used to produce adenovirus mutants. "Adenovirus mutants" mean 10 adenoviruses which have at least one mutation in the ElA and/or ElB genes. In a preferred embodiment, they do not, however, in contrast to adenoviral gene transfer vectors, harbor any therapeutic genes. A typical example thereof comprises adenovirus mutants in 15 which the ElB 55 kD protein is not expressed (for example the adenovirus mutant d11520 (Barker et al., Virology 156, 107-121, 1987)) . Adenovirus mutants in which the ElB 55 kD protein is not expressed are of great interest for the therapy of oncoses because the 20 virus mutant replicates exclusively in tumor cells and not or to a negligible extent in primary normal cells (Bischoff et al., Science 274, 373-376, 1996; Kirn et al., Nature Med. 4, 1341-1342, 1998). A preferred embodiment is the use of a 25 permanent amniocytic cell line for producing adenovirus vectors, AAV (adeno-associated virus) vectors, retrovirus vectors, lentivirus vectors, chimeric adenovirus-AAV vectors, chimeric adenovirus-retrovirus vectors and/or chimeric adenovirus-lentivirus vectors. 30 A use for producing herpes vectors is also possible. AAV vectors normally comprise only the ITRs of AAV and some adjacent, noncoding AAV sequences. Their capacity for uptake of foreign DNA is about 4.5 kb. As described above, various systems exist for producing 35 recombinant AAV vectors. It is common to all these systems that the components necessary for replication, expression and packaging of the recombinant vector are provided. Specifically, these comprise expression - 25 cassettes which code for the AAV rep and cap proteins, and the adenoviral helper functions. The adenoviral helper functions necessary for AAV production are the ElA, ElB, E2, E4 and VA genes. The ElA and ElB 5 functions are provided in the ElA- and ElB-expressing amniocytic cell lines and can therefore be used to produce AAV vectors. The E2, E4 and VA functions can be provided by coinfection with adenovirus or by cotransfection with E2-, E4- and VA-expressing plasmids 10 or by using adenovirus/AAV or herpes simplex virus/AAV hybrid vectors (Samulski et al., supra; Allen et al., supra; Tamayose et al., supra; Flotte et al., supra; Conway et al., supra; Chiorini et al., supra; Ferrari et al., supra; Salvetti et al., supra; Xiao et al., 15 supra; Grimm et al., supra; Zhang et al., supra). Retrovirus vectors, that is to say vectors derived from retroviruses, are likewise of great importance as vehicles for transfection within the scope of gene therapeutic procedures, for example for 20 gene therapy in-- the central nervous system (Suhr et al., Arch. Neurol. 56, 287-292, 1999). Retroviral vectors can be produced in stable vector-producing cell lines or by transient transfection. The individual components used to produce retroviral vectors normally 25 include one or more plasmids which express the structural proteins and the replication and integration proteins, as well as a plasmid which comprises the vector itself (Miller, in:. Retroviruses, Coffin, Hughes, Varmus ed., Cold Spring Harbor Laboratory 30 Press, 1997, pp. 437-473). If those plasmids which contain an origin of replication, such as, for example, the SV40 origin of replication, are used, the amniocytic cell lines are modified so that proteins which promote replication of the plasmid are stably 35 expressed. For example, in the case of plasmids which contain the SV40 origin of replication, an amniocytic cell line which expresses the T antigen of SV40 is used.

- 26 Lentivirus vectors are vectors derived from lentiviruses (Naldini et al., Science 272, 263-267, 1996; Dull et al., J. Virol. 72, 8463-8471, 1998). Lentiviral vectors can be produced in stable vector 5 producing cell lines or by transient transfection. "Chimeric vectors" mean vectors which are the product of a fusion of nucleic acids from two or more different viral vectors. Permanent amniocytic cell lines can be used according to the present description 10 for producing chimeric vectors. In this system, for example, an adenovirus vector, preferably an adenovirus vector of large capacity, harbors a DNA fragment which [lacuna] the sequence information for an integrating virus which is derived, for example, from a retrovirus 15 or from AAV. After transcription of a target cell, the integrating virus harboring a therapeutic gene is released from the adenoviral background (for example in the case of a retroviral insert by producing infectious retroviral particles which transduce neighboring cells 20 and integrate stably as DNA). Examples of chimeric vectors produced in 293 cells have been described in the past, for example as chimeric adenovirus-retrovirus vectors (Feng et al., Nature Biotech. 15, 866-870, 1997) and as chimeric adenovirus-AAV vectors (Recchia 25 et al., Proc. Natl. Acad. Sci. USA 96, 2615-2620, 1999). Production in ElA and ElB-expressing amniocytic cell lines is to be preferred because, in contrast to 293 cells, replication-competent vectors cannot be generated by homologous recombination. 30 Adenovirus vectors, that is to say vectors derived from adenoviruses, are of great importance in particular as vehicles for transfection within the scope of gene therapeutic procedures. The adenovirus vectors may be first-generation adenovirus vectors, 35 second-generation adenovirus vectors, adenovirus vectors of large DNA capacity and/or deleted adenovirus vectors, which are produced with the aid of a permanent amniocytic cell line.

- 27 a) Production of first-generation adenoviral vectors First-generation adenoviral vectors are usually characterized by deletions of the ElA and ElB genes. Some first-generation adenoviral vectors comprise, in 5 addition to the deletion of the ElA and ElB genes, also deletions of the E3 region. E3 functions are dispensable for the growth of adenoviral vectors in cell culture. First-generation adenoviral vectors can be 10 produced in E1A- and ElB-expressing amniocytic cell lines. This is done by infecting the E1A- and ElB-expressing cells preferably with 3-5 infectious units per cell (3-5 MOI). After about 36 to 72 hours, the cells show a cytopathic effect. The cells are 15 harvested by standard protocols. Adenoviral vector can be purified from them by CsCl density gradient centrifugation or by chromatographic processes. b) Production of second-generation adenoviral vectors 20 Second-generation adenoviral vectors are characterized by deletions of ElA and ElB genes. Some second-generation adenoviral vectors also comprise a deletion of the E3 region. In addition to the deletion of the ElA and ElB genes, second-generation adenoviral 25 vectors are characterized by inactivation and preferably deletion of at least one other essential adenoviral gene, for example an E2A gene, an E2B gene and/or a E4 gene, or, for example, by deletions of E2 functions in combination with deletions of E4 30 functions. To produce second-generation adenoviral vectors, the functions which the vector itself does not express, due to inactivation and/or deletion, must be provided by the amniocytic cell line. For this purpose, 35 it is possible for amniocytic cell lines which stably express ElA and ElB to be stably modified by transfection of expression cassettes which express the gene products coding for one or more other adenoviral - 28 functions. For example, to produce a second-generation adenoviral vector which has, in addition to the deletion of the ElA and ElB genes, also a deletion of an E2A, E2B and/or E4 gene, the appropriate gene or 5 genes is (are) introduced by transfection together with a selection marker into the ElA- and ElB-expressing amniocytic cell line. Cell clones which, in addition to the expression of ElA and ElB functions, also express E2A, E2B and/or E4 functions can then be used to 10 produce the particular second-generation vector. The E2 and/or E4 genes are usually under the transcriptional control of a heterologous promoter, which either is constitutively active or can be regulated. 15 c) Production of adenoviral vectors of large DNA capacity Adenoviral vectors of large DNA capacity are characterized by deletion of most or all of the viral coding sequences. These vectors preferably comprise 20 only the viral ITRs and the viral packaging signal. The adenoviral functions are provided by a helper virus in trans. Various systems for producing adenoviral vectors of large DNA capacity have been described. It is common to all the systems described to date and using a helper 25 virus that the helper virus corresponds to a replication-deficient, E1A- and ElB-deleted adenovirus. The helper virus comprises either a complete packaging signal (Mitani et al., Proc. Natl. Acad. Sci. USA 92, 3854-3858, 1995; Fisher et al., Virology 217, 11-22, 30 1996; Kumar-Singh and Chamberlain, Hum. Mol. Genet. 5, 913-921, 1996) or a mutated packaging signal (Kochanek et al., Proc. Natl. Acad. Sci. U.S.A. 93, 5731-5736, 1996) . In the latter case, the vector is preferably packaged in viral capsids because the helper virus 35 contains an attenuated packaging signal and therefore is packaged less efficiently. Alternatively, the packaging signal of the helper virus can be excised after the infection of the producer cell line by using - 29 a recombinase (Parks et al., Proc. Natl. Acad. Sci. USA 93, 13565-13570, 1996; Hardy et al., J. Virol. 71, 1842-1849, 1997) . For example, the packaging signal of the helper virus can be flanked by loxP recognition 5 sequences of bacteriophage P1. Expression of the Cre recombinase of bacteriophage P1 results in excision of the packaging signal of the helper virus. However, because of the absence of the packaging signal, no packaging of the helper virus into capsids takes place. 10 The Cre recombinase gene of bacteriophage P1 is introduced by transfection together with a selection marker into the E1A- and ElB-expressing amniocytic cell line. Cell clones which, in addition to expression of ElA and ElB functions, also express the Cre function of 15 bacteriophage P1 can then be used to produce the particular vector of large DNA capacity. d) Production of "deleted" adenoviral vectors "Deleted" adenoviral vectors have been 20 described as first-generation vectors which have loxP recognition sequences of bacteriophage P1 positioned in the viral genome in such a way that, on infection of Cre-expressing 293 cells, most of the viral coding sequences or all the viral coding sequences are deleted 25 by recombination between the loxP recognition sequences. The genome size of these vectors is about 9 kb. The capacity for uptake of foreign DNA is likewise about 9 kb (Lieber et al., J. Virol, 70, 8944-8960, 1996). For use in the production of deleted 30 adenoviral vectors, the Cre recombinase gene of bacteriophage P1 is introduced by transfection together with a selection marker into the E1A- and ElB expressing amniocytic cell line. Cell clones which, in addition to expression of ElA and ElB functions, also 35 express the Cre function of bacteriophage P1 can then be used to produce the particular deleted Ad vector.

- 30 e) Production of tropism-modified gene transfer vectors In a preferred embodiment, the permanent amniocytic cell line is used to produce tropism 5 modified gene transfer vectors. The tropism of a virus and of a viral vector derived from this virus decides whether a particular cell type can be successfully transduced with a vector or not. Uptake of a gene transfer vector into a cell is the first step for 10 successful gene transfer into this cell. The tropism of a viral vector is thus an essential factor for efficient in vitro or in vivo gene transfer into a particular cell or into a tissue. Interaction of the surface of a viral vector (of the capsid in the case of 15 adenoviral or AAV vectors, of the virus envelope in the case of retroviral or lentiviral vectors) with the cell membrane of a target cell is necessary for uptake into a particular cell. Although the exact mechanism of uptake of a viral vector into a target cell sometimes 20 varies between different vectors, in all cases the interaction of surface structures of the viral vector (usually protein ligands) with structures on the target cell (usually receptors or adhesion molecules) plays an essential part. Uptake of adenoviral vectors takes 25 place, for example, by receptor-mediated endocytosis. This entails parts of the adenoviral capsid binding to cellular receptors. In the case of adenoviral vectors derived from Ad2 or Ad5, according to the current state of knowledge there is usually binding of part of the 30 knob domain of the fiber proteins to the coxsackie adenovirus receptor (CAR) and part of the penton base to avP3 or avP5 integrins. The binding of the knob domain on CAR is, according to the current state of knowledge, necessary for adhesion of the vector to the 35 cell membrane of the target cell, whereas binding of the penton base to integrins is necessary for internalization of the vector into the target cell.

- 31 Amniocytic cell lines can be used to produce tropism-modified vectors. This applies, for example, to the production of first- and second-generation adenoviral vectors, to adenoviral vectors of large DNA 5 capacity, to deleted adenoviral vectors, to chimeric adenoviral vectors, to AAV vectors, to retroviral and/or lentiviral vectors. Various strategies can be used to produce tropism-modified vectors in amniocytic cell lines. The strategy used for the particular 10 tropism modification may vary for different vectors (for example adenoviral vector, AAV vector, retroviral vector) . It is common to the various strategies that the surface of the particular vector (virus capsid in the case of adenoviral and AAV vectors, virus envelope 15 in the case of retroviral and lentiviral vectors) is altered so that the binding of the vector to the target cell is altered. Examples of modifications for adenoviral vectors are: 20 a) Exchange of fiber proteins between different serotypes: this results in adenoviral vectors whose capsid carries a fiber protein of a different serotype. Examples thereof, are exchange of the natural fiber protein of adenoviral vectors 25 derived from serotype 2 by a fiber protein derived from serotype 17 (Zabner et al., J. Virol. 73, 8689-8695, 1999) or from serotype 9 (Roelvink et al., J. Virol. 70, 7614-7621, 1996). Other examples are exchange of the natural fiber protein 30 of adenoviral vectors derived from serotype 5 by a fiber protein derived from serotype 7a (Gall et al., J. Virol, 70, 2116-2123, 1996) or from serotype 3 (Stevenson et al., J. Virol. 71, 4782-4790, 1997; Krasnykh et al., J. Virol. 70, 35 6839-6846, 1996; Douglas et al., Neuromuscul. Disord. 7, 284-298, 1997).

- 32 b) Removal of the fiber protein: the fiber protein can be removed by processes of genetic manipulation so that uptake of the vector takes place solely via interaction of the penton base or 5 of the hexon protein (Falgout et al., J. Virol. 62, 622-625, 1988; Legrand et al., J. Virol. 73, 907-919, 1999). c) Modification of the C terminus of the fiber 10 protein with a peptide: example thereof are modification of the C terminus with a polylysine peptide (Yoshida et al., Hum. Gene Ther. 9, 2503-2515, 1998: Wickham et al., Nat. Biotechnol. 14, 1570-1573, 1996; Wickham et al., J. Virol. 71, 15 8221-8229, 1997), a polyhistidine peptide (Douglas et al., Nat. Biotechnol. 17, 470-475, 1999) or a gastrin-releasing peptide (Michael et al., Gene Ther. 2, 660-668, 1995). 20 d) Modification of parts of the knob domain of the fiber protein by insertion of a peptide: examples thereof are insertion of a FLAG epitope (Krasnykh et al., J. Virol. 72, 1844-1852, 1998) or insertion of an RGD peptide (Dmitriev et al., 25 J. Virol. 72, 9706-9713, 1998; Kasono et al., Clin Cancer Res. 5, 2571-2579, 1999). e) Modification -of the penton base: one example thereof is replacement of an RGD motif within the 30 penton base by an LDV motif with the aim of mediating binding of the vector to u4p1 integrins (Wickham et al., Gene Ther. 2, 750-756, 1995). f) Modification of the hexon protein: one example 35 thereof is insertion of an epitope derived from poliovirus type 3 (Crompton et al., J. Gen. Virol. 75, 133-139, 1994).

- 33 An alternative strategy which can be used to alter the tropism of vectors produced in amniocytic cell lines is based on the use of ligands which mediate binding of the vector to cell membrane structures such 5 as, for example, cellular receptors or adhesion molecules. These ligands may be peptides, proteins or else antibodies. The ligands can be linked to the surface of the vectors by various processes. The linkage of the ligands to the surface of the vectors 10 (of the capsids in the case of adenoviral or AAV vectors) can be produced by using antibodies or by a chemical crosslinking reaction. On use of antibodies it is possible to use antibodies whose specificity is directed against the capsid of the vector (for example 15 against the knob domain of the fiber protein). Alternatively, it is possible to use antibodies whose specificity is directed against an epitope which has been introduced as neoepitope (for example a FLAG epitope or a myc epitope) into the capsid of the 20 vector. Examples thereof are well known to the skilled worker. Examples of the use of bispecific antibodies are described in Wickham et al., J. Virol. 70, 6831-6838, 1996 (anti-FLAG/anti-a-integrin) ; in Wickham et al., Cancer Immunol. Immunther. 45, 149-151, 1997; 25 Harari et al., Gene Ther. 6, 801-807, 1999 (anti FLAG/anti-E-selectin) for transduction of endothelial cells; in Miller et al., Cancer Res. 58, 5738-5748, 1998; Blackwell et al., Arch. Otolaryngol. Head Neck Surg. 125, 856-863, 1999 (anti-Ad/anti-EGFR) for 30 transduction of tumor cells; in Wickham et al., J. Virol. 71, 7663-7669, 1997 (anti-FLAG/anti-CD3) for transduction of T cells; in Tillman et al., J. Immunol. 162, 6378-6383, 1999 (anti-CD40/anti-Ad) for transduction of dendritic cells. Examples of the use of 35 single-chain antibodies with specificity for one virus capsid determinant which is coupled to a ligand are described in Watkins et al., Gene Ther. 4, 1004-1012, 1997; in Goldman et al., Cancer Res. 57, 1447-1451, - 34 1997; Rancourt et al., Clin. Cancer Res. 4, 2455-2461, 1998; Gu et al., Cancer Res. 59, 2608-2614, 1999; Rogers et al., Gene Ther. 4, 1387-1392, 1997 (anti Ad/FGF2) for transduction of FGF2-receptor-expressing 5 tumor cells; in Douglas et al., Nat. Biotechnol. 14, 1574-1578, 1996; Douglas et al., Neuromuscular Disord. 7, 284-298, 1997 (anti-Ad/Folat) for transduction of tumor cells which express the folic acid receptor on the cell surface. 10 In the case of gene transfer vectors in which the natural tropism has been abolished and replaced by another tropism, for example by introducing a ligand into the knob domain of the fiber protein of Ad5, it may be necessary to modify a permanent amniocytic cell 15 line by the preferably stable expression of a receptor which recognizes this new ligand (Douglas et al., Nat. Biotechnol. 17, 470-475, 1999). It is likewise possible for the permanent amniocytic cell line to be used to produce gene transfer vectors which have a defect in 20 the production of one or more structural proteins. This is done by complementing the particular defects of the gene transfer vector in the permanent amniocytic cell line. For example, an adenoviral vector which has a mutation in the gene coding for the fiber protein can 25 be produced in an amniocytic cell line which complements the defect in the fiber protein. This is achieved by introducing a fiber expression cassette into the amniocytic cell line and stable or inducible expression of the fiber protein in this amniocytic cell 30 line (Von Seggern et al., J. Gen. Virol. 79, 1461-1468, 1998). The fiber protein expressed in the amniocytic cell line may be a natural, unmodified fiber protein or else an altered, for example tropism-modified, fiber protein (Von Seggern et al., supra) . It is also 35 possible to produce adenoviral vectors completely lacking the fiber protein in the permanent amniocytic cell line (Legrand et al., J. Virol., 73, 907-919, - 35 1999; Von Seggern et al., J. Virol. 73, 1601-1608, 1999). The use of E1A- and ElB-expressing amniocytic cell lines is to be preferred because, in contrast to 5 293 cells, no generation of replication-competent vectors can take place by homologous recombination. In a particular embodiment of the aspect of the use of an amniocytic cell line for producing gene transfer vectors, this cell line is the cell line according to 10 the invention. Therapeutic genes The genes, in particular the therapeutic genes [sic], which can be encoded and expressed by vectors 15 produced in transformed amniotic cells, that is to say a permanent amniotic cell line, can be, for example, any muscle proteins, coagulation factors, membrane proteins or cell cycle proteins. Examples of proteins which can be expressed by vectors produced in 20 transformed amniocytes are dystrophin (Hoffman et al., Cell 51, 919, 1987, factor VIII (Wion et al., Nature 317, 726 1985, cystic fibrosis transmembrane regulator protein (CFTR) (Anderson et al., Science 251, 679, 1991, ornithine transcarbamylase (OTC) (Murakami et 25 al., J. Biol. Chem., 263, 18437, 1988, alphal-antitrypsin (Fagerhol et al., in: Hum. Genet., vol. 11, Harris ed., Plenum, New York, p. 1, 1981. The genes coding for proteins are known and can be cloned from genomic or cDNA banks. Examples of such genes are 30 the dystrophin gene (Lee et al., Nature 349, 334, 1991, the factor VIII gene (Toole et al., Nature 312, 342 91984 [sic]), the CFTR gene (Rommens et al., Science 245, 1059, 1989, Riordan et al., Science 245, 1066, 1989, the OTC gene (Horwich et al., Science 224, 1066, 35 1984, and the alphal-antitrypsin gene (Lemarchand et al., Proc. Natl. Acad. Sci. USA, 89, 6482, 1992. Examples of other genes expressed by vectors which can be produced in transformed amniocytes are the - 36 p53 gene for treating oncoses (Wills et al., Hum. Gene Ther. 5, 1079, 1994, Clayman et al., Cancer Res. 55, 1, 1995, the Rb gene for treating vascular proliferative disorders (Chang et al., Science 267, 518, 1995, or the 5 thymidine kinase gene of herpes simplex virus (HSV) type 1 for the therapy of oncoses. The gene expressed by vectors produced in transformed amniocytes does not necessarily code for a protein. Thus, for example, it is possible for functional RNAs to be expressed. 10 Examples of such RNAs are antisense RNAs (Magrath, Ann. Oncol. 5, Suppl 1), 67-70 1994, Milligan et al., Ann. NY Acad. Sci. 716, 228-241, 1994, Schreier, Pharma. Acta. Helv., 68, 145-159 (1994), and catalytic RNAs (Cech, Biochem. Soc. Trans. 21, 229-234, 1993; Cech, 15 Gene 135, 33-36, 1993; Long et al., FASEB J. 7, 25-30, 1993; Rosi et al., Pharm. Therap. 50, 245-254, 1991). Vectors produced in transformed amniocytes may, in addition to the therapeutic gene, comprise any reporter gene in order to be able to follow expression 20 of the vector better. Examples of reporter genes are known in the prior art and include, for example, the P-galactosidase gene (Fowler et al., Proc. Natl. Acad. Sci. USA 74, 1507, 1977). Vectors which can be produced in transformed 25 amniocytes may comprise more than a single gene. The maximum number of genes which can be produced in such vectors depends on the uptake capacity of the particular vector and on the size of the genes. The choice of the promoters which control 30 expression of the therapeutic genes of vectors produced in transformed amniocytes is not critical. Viral or nonviral promoters which show constitutive, tissue specific or regulable activity can be used for expressing a protein or a functional RNA. The SV40 or 35 cytomegalovirus promoter (Andersson et al., J. Biol. Chem. 264, 8222-8229, 1964) can be used, for example, for constitutive expression of a gene. The use of the muscle creatine kinase (MCK) promoter permits tissue- - 37 specific expression of a protein or of a functional RNA in skeletal muscle and myocardium. Gene expression can be controlled quantitatively and qualitatively by the use of a regulable system (Furth et al., Proc. Natl. 5 Acad. Sci. USA 91, 9302-9306, 1992 [sic]). It is possible to include in vectors which can be produced in transformed amniocytes genetic elements which influence the behavior of the vector inside the recipient cell. Examples of such elements are elements 10 which facilitate nuclear targeting of the vector DNA (Hodgson, Biotechnology 13, 222-225, 1995). Vectors produced in this way can be used in vitro or in vivo. An in vitro gene transfer takes place outside the body, for example by adding the vector to 15 cells in culture or to primary cells which have been taken from the body for the purpose of gene transfer. In the case of in vivo gene transfer, vector particles can be applied in various ways depending on the tissue which is to be transduced. Examples are injection into 20 the arterial or venous vascular system, direct injection into the relevant tissue (for example liver, brain, muscle), instillation into the relevant organ (for example lung or gastrointestinal tract) or direct application onto a surface (for example skin or 25 bladder). The following figures and example are intended to illustrate the invention in detail without restricting it thereto. 30 DESCRIPTION OF THE FIGURES Fig. 1 shows a summary of the clonings: Fig 1A depicts diagrammatically the cloning steps for plasmid STK146. Fig. 1B depicts the left-terminal 35 approx. 15% of the genome of adenovirus type 5, including the El RNAs, the coding regions, the starting points of ElA and ElB transcription, and the splice donor and splice acceptor sites - 38 and polyadenylation sequences which are important for the cloning. It is important that the splice donor site at base pair 3511 of Ad5 has been retained in the cloning for plasmid 5 STK146, but that the splice acceptor site and the polyadenylation signal have been replaced by corresponding functions of SV40. In addition, the ElA promoter of Ad5 has been replaced by the PGK promoter. Thus STK146 10 contains the Ad5 sequences from base pair 505-5322 and the cell lines transformed with this plasmid contain no Ad5 sequences which are present in first- or second-generation adenoviral vectors or in loxP helper viruses. 15 Fig. 2 shows the cell islets (Fig. 2B and Fig. 2B) obtained from amniocytes by transformation by adenoviral El functions, and the cell lines N52.E6 (DSM ACC2416; Fig. 2C) and N52.F4 (Fig. 20 2D) cloned from single cells. This time it should be noted that the cell lines and single cell clones differ morphologically from the amniocytes in that they are usually smaller and there is no contact inhibition of their growth. 25 Fig. 3 shows the integration status of STK146 in eight different El-transformed amniocytic cell lines by means of a Southern blot. 30 Fig. 4 shows, listed in a table, the production of a first-generation adenoviral vector in various cloned cell lines on the basis of bfu (blue forming units) per cell and the efficiency of transfection of the appropriate cell lines on 35 the basis of plaque formation.

- 39 Fig. 5 shows the expression of the ElA and ElB proteins of Ad5 in eleven cloned amniocytic cell lines (Western blot). 5 Fig. 6 shows the time course of the synthesis of recombinant adenoviral vectors in two cloned cell lines N52.E6 and N52.F4. Fig. 6A shows the synthesis of a first-generation adenoviral vector, and Fig. 6B shows the synthesis of an 10 adenoviral vector with large DNA capacity. EXAMPLES 1. Clonings 15 Fig. 1 depicts a summary of the clonings. a) Plasmid STK136 20 Plasmid STK 136 contains the murine phosphoglycerate kinase promoter (seq. No. 1; Adra et al., Gene 60, 65-74, 1987) in pBluescript KsII (Stratagene) and was produced as follows: 3.5 pg of plasmid PGK-hAAT (Kay et al., 25 Hepatology 21, 815-819, 1995) were digested with EcoRV and fractionated by size in a 1.5% agarose gel. The 0.5 kb band containing the PGK promoter fragment which was sought was, after staining in ethidium bromide, cut out and the DNA was electroeluted. At the same time, 30 pBluescript KSII was digested with EcoRV and HincII, and the free DNA ends were dephosphorylated. After subsequent phenol/chloroform extraction and ethanol precipitation, equimolar amounts of these DNA fragments were ligated and transformed into ultracompetent XL-2 35 Blue bacteria (Stratagene). The plasmid clones were characterized by means of a restriction digestion, and the plasmid resulting therefrom was called STK136 (isolate #6).

- 40 b) Plasmid STK137 Plasmid STK137 contains the complete El expression cassette of Ad5 including the 3' splice and 5 polyadenylation signals from SV40 and was produced as follows: PCR amplification of the Ad5 sequence bp 505-841 (PCR I) (Seq. No. 2) 10 10 ng of the plasmid pXC1 (Microbix) were amplified together with 400 ng each of the oligonucleotides 27759 (Seq. No. 3) and 27634 (Seq. No. 4), 0.2 mM dNTPs and 1.25 U Pfu polymerase in 10 mM KCl, 10 mM (NH 4

)

2

SO

4 , 20 mM Tris/HCl, pH 8.75, 15 2 mM MgSO 4 , 0.1% Triton X-100, 100 pg/ml BSA under the following conditions: I 10 minutes at 944C, II 1 minute at 94*C, 20 2 minutes at 50*C, 3 minutes at 72 0 C, III 10 minutes at 72 0 C. repeating step II in 15 cycles. The DNA was purified 25 using the QIAquick PCR purification kit (Qiagen) as stated by the manufacturer and was precipitated with ethanol. To clone the PCR fragment, 2.5 pLg of pBluescript KSII were digested with EcoRV, and the free 30 DNA ends were dephosphorylated, ligated in equimolar amounts with the PCR fragment and transformed into XL-2 Blue cells. The plasmid resulting therefrom is referred to as #1 hereinafter. 35 PCR amplification of the Ad5 sequence bp 3328-3522 (PCR II) Seq. No. 5): 10 ng of the plasmid pXC1 (Microbix) were amplified together with 400 ng each of the - 41 oligonucleotides 27635 (Seq. No. 6) and 27636 (Seq. No. 7) under the conditions described above. After the PCR reaction, the DNA was extracted with phenol/chloroform, precipitated with ethanol, digested with EcoRI, again 5 extracted with phenol/chloroform, precipitated and dissolved in 30 pl of TE. PCR amplification of the 3' splice and polyadenylation signal from SV40 with the aid of the plasmid pGL2-Basic 10 bp 1978-2749 (PCR II) (seq. no. 8): 20 ng of the plasmid pGL2-Basic (Promega, GenBank/EMBL Acc. No.: X65323) were amplified together with 800 ng each of the oligonucleotides 27637 (seq. No. 9) and 27638 (seq. No. 10), 0.4 mM dNTPS and 15 2.5 U of Pfu polymerase under the conditions described above. After the PCR reaction, the DNA was extracted with phenol/chloroform, precipitated with ethanol, digested with EcoRI, again extracted with phenol/chloroform, precipitated in ethanol and 20 dissolved in 30 pl of TE. Then 10 pl each of DNA from PCR II and III were ligated in a volume of 50 pl, extracted with phenol/chloroform, precipitated with ethanol and digested with BamHI in a volume of 100 pl. After renewed phenol/chloroform extraction and ethanol 25 precipitation, the DNA was ligated with equimolar amounts of pBluescript KSII DNA which had previously been digested with BamHI and dephosphoylated. The plasmid resulting therefrom is referred to as #29 hereinafter. For further cloning, 3.5 pg of plasmid DNA 30 #29 were digested with SacII and BglII, dephosphorylated, extracted with phenol/chloroform and precipitated in ethanol. At the same time, 3.5 pg of pXC1 were digested with BglII and SacII, and the 2.9 kb fragment was fractionated by electrophoresis and 35 electroeluted. Equimolar amounts of the two DNAs were ligated and transformed into XL-2 Blue cells. The plasmid resulting therefrom is referred to as #5 hereinafter. For the final cloning of STK137, plasmid - 42 #1 was digested with HincII and BspEI and fractionated by electrophoresis, and an approx. 350 bp fragment was electroeluted. As vector DNA, plasmid #5 was digested with KspI (isoschizomer of SacII), the ends were filled 5 in with T4 polymerase, and phenol/chloroform extraction and ethanol precipitation were carried out. The DNA was then digested with BspEI, the ends were dephosphoylated, and phenol/chloroform extraction and ethanol precipitation were again carried out. The two 10 DNAs were ligated and transformed into XL-2 Blue cells. The plasmid resulting therefrom was called STK137 (isolat #34). c) Plasmid STK146 (Seq. No. 18) 15 Plasmid STK146 contains the murine PGK promoter, the complete El region of Ad5 (bp 505-3522) and the 3' splice and polyadenlylation signal of SV40. For the cloning, 4 pLg of STK137 were digested with EcoRV and BamHI and fractionated by 20 electrophoresis, and the 3.7 kb fragment was electroeluted. In addition, 3.3 pg of STK136 were digested with EcoRV and BamHI, dephosphorylated, phenol/chloroform extracted and precipitated with ethanol. Equimolar amounts of the two plasmids were 25 ligated and transformed into XL-2 Blue cells. The plasmid resulting therefrom was called STK139. Final sequence analysis of STK139 revealed a mutation at the bp 2613 (the numbering in this connection refers to the Ads DNA sequence), which led to a tyrosine to 30 asparagine amino acid exchange (2613 TAC -> GAC). For this reason, the fragment containing the mutation in STK139 was replaced by the BstEII (bp 1915) -BglII (bp 3328) fragment from pXC1. This was done by digestion of STK139 with BstEII and BglII, 35 dephosphoylation, phenol/chloroform extraction, ethanol precipitation, fractionation by electrophoresis and electroelution of the 5.8 kb fragment. pXC1 was likewise digested with BstEII and BglII and - 43 fractionated by electrophoresis, and the 1.4 kb fragment was electroeluted. After ligation and transformation, DNA from 4 plasmid clones was sequenced; two of them contained the correct sequence 5 at bp 2613. Isolate number 2 was sequenced completely and is referred to as STK146 hereafter. d) Sequence analysis of STK146 10 500 ng of STK146 #2 were sequenced with 10 pmol of the following sequence primers under standard conditions: Primer 28231 Ads nt. 901-920 (Seq. No. 11) 28232 Ad5 nt. 1301-1320 (Seq. No. 12) 15 28233 Ad5 nt. 1701-1720 (Seq. No. 13) 28234 Ad5 nt. 2100-2119 (Seq. No. 14) 28235 Ad5 nt. 2500-2519 (Seq. No. 15) 28236 Ad5 nt. 2853-2872 (Seq. No. 16) 28237 Ad5 nt. 3249-3268 (Seq. No. 17) 20 2. Cultivation of primary anniocytes and cell lines All the cell culture reagents, media and sera were purchased from GIBCO Life Technologies. The cell line 293 which was used as control in some experiments 25 was cultivated in modified Eagle's medium (MEM) with 10% fetal calf serum (FCS) , 1x penicillin/streptomycin at 37 0 C (100x, Cat# 10378-016), 95% humidity and 5%

CO

2 . The new El-transformed cell lines were produced using primary fetal cells which had been obtained from 30 amniotic fluid by amniocentesis as part of prenatal diagnosis. After the biopsy, the cells were seeded by routine methods in plastic culture bottles and cultivated in Ham's F10 medium (nutrient mixture Ham's F10 with L-glutamine, Cat# 31550-023), 10% FCS, 2% 35 Ultroser@ G, 1x antiobiotic/antimycotic solution (100x, Cat# 15254-012), 2.5 pg/ml Fungizione@ (amphotericin B, Cat# 15290-018). Some of the cells adhered to the base of the cell culture bottle and proliferated. Sufficient - 44 cells for a chromosome analysis were available after about 2 weeks. After establishment of the karyotype, amniocyte cultures with numerically and structurally normal chromosomes were used to produce the cell line. 5 Cells from three different sources, taken by amniocentesis either 3, 6 or 7 weeks beforehand, were used in various experiments. The nutrient medium used was Ham's F10 medium, 10% FCS, 2% Ultroser@Cr, 1x antibiotic/antimycotic solution, 2.5 pig/ml Fungizione@. 10 The culture conditions were 370C, 95% humidity and 5%

CO

2 . Seven days after transfection, the amniocytes were cultivated further in Ham's F10 medium, 10% FCS, 1x penicillin/streptomycin. After the generation of single-cell clones, these were transferred to new 15 dishes and cultivated further in alpha-MEM with 10% FCS, 1x penicillin/streptomycin. 3. Transfection and transformation of amniocytes 20 For the transfection, the amniocytes were seeded on cell culture dishes (diameter 60 mm, surface area 22.1 cm 2 ) at a density of 2 - 5 x 10s per dish and transfected the following day. For the transfection, 20 tg of plasmid STK146 were digested with ScaI, 25 extracted with phenol/chloroform, precipitated with ethanol and taken up in 20 pl of TE, which gave a DNA concentration of 0.5 pg/pl. In the initial experiments, amniocytes were transfected 3 or 7 weeks after removal, in 5 dishes each, with the Effectene transfection kit 30 as stated by the manufacturer (Qiagen) as follows: 4 pl of STK146 digested with ScaI were mixed with 146 pl of EC buffer. After addition of 8 pl of enhancer, the solution was briefly vortexed and incubated at room temperature for 5 minutes. Then 25 pl 35 of Effectene were added and, after vortexing for 10 seconds, incubated at room temperature for a further 10 minutes. During this, the medium was cautiously aspirated off the cells and replaced by 4 ml of fresh - 45 nutrient medium (see Section 2. above). After the incubation was complete, the transfection mixture was mixed with 1 ml of fresh nutrient medium and cautiously added dropwise to the cells. The cells were cultivated 5 further as described above. Seven days after the transfection, the cells in each dish were transferred to a larger dish (diameter 150 mm, surface area 147.8 cm 2 ). This was done by cautiously aspirating off the medium, and the cells being washed with PBS, 10 detached in trypsin and transferred to a new dish and cultivated further as described in Section 2. 18 to 22 days after the transfection clonal cell islets were clearly to be seen and were clearly distinguished morphologically from the amniotic cells (Figs. 2A, 2B). 15 The main proportion in an untransformed amniocyte culture consists of larger cells which show contact inhibition of their growth. Cells in transformed single-cell colonies are very much smaller, grow much more quickly and show no contact inhibition of their 20 growth. They grow as cell islets consisting of smaller cells which are crowded tightly together, and are unambiguous under the light microscope and can be identified without difficulty. These cell islets were picked and transferred to a new dish (diameter 60 mm) 25 containing the medium described above. After further growth, the cell lines were transferred to 147.8 cm 2 cell culture dishes and cultivated further as described under 2. After the first transfer to 147.8 cm 2 cell culture dishes, the cell passages were counted. 30 Initially, about 40 cell clones from the cells which had been transfected 3 and 7 weeks after removal were cultivated further. Subsequently, that is to say after prolonged cultivation, there was a drastic change in the morphology of some of the cell clones, and they 35 showed instability in their growth characteristics. The further experiments were restricted to further cultivation and analysis of eight morphologically stable cell lines. These were referred to as follows: - 46 GS.A55 (produced from amniocytes transfected 3 weeks after removal), GS.N21, GS.N24, GS.N27, GS.N49, GS.N51, GS.N52, GS.N53 (produced from amniocytes transfected seven weeks after removal). 5 During the first passages all the cell clones showed a comparable morphology, but this was changed by subsequent passages. Thus, for example, some cell clones changed to assume a highly rounded shape and, after further passages, they were no longer adherent. 10 Other cell clones showed extensive vacuolization, but this did not appear to have any effect on their growth. After the single-cell cloning, all the cell lines showed a uniform morphology and, for example, N52.EG and N52.F4 had an epithelial appearance. They were 15 comparable to 293 cells in their growth rate and cell density. 4. Efficiency of the transformation In order to determine the efficiency of the 20 transformation by the El functions more accurately, seven new dishes each containing 2-5 x 105 cells were transfected as described in Section 3. The cells were transferred only 24 hours after the transfection to dishes 147.8 cm 2 in size and were cultivated further in 25 Ham's F-10 medium, 10% fetal calf serum, 2% Ultroser@ G, 1 x antiobiotic/antimyotic solution, 2.5 pg/ml Fungizione for 5 days and in Ham's medium, 10% fetal calf serum, 1x penicillin/streptomycin solution for a further 25 days. A dish with untransfected cells was 30 cultivated under the same conditions as a control. During this time, the morphologically clearly distinguishable (see Fig. 2A, B) colonies resulting from single transformation events were counted. Single cell clones could be counted on all the cell culture 35 dishes apart from the untransfected control dish. On average there were 4 cell clones per plate, which corresponded to a transformation efficiency of 1 in 0.5-1 x 105 cells.

- 47 5. Single-cell cloning As already mentioned, some of the cell lines showed different morphological characteristics, which 5 is why up to ten single cell lines were set up from each cell line. The passages for the individual cell lines differed in these cases: GS.A55: P17, GS.N21: P24, GS.N24: P20, GS.N27: P19, GS.N49: P21, GS.N51: P39, GS.N52: P22, GS.N53: P20. For this purpose, the 10 cells were detached from the cell culture dishes and, at a concentration of 5 x 106 cells/ml, diluted 1:1000, 1:50,000 and 1:500,000 in nutrient medium. 100 pl cells from all the dilutions were seeded onto 96-well plates, and the cell clones which had unambiguously resulted 15 from single cells were cultivated further. Fig. 2C shows the cell line GS.N52.E6 (DMSZ [sic] No.), and Fig. 2D shows cell line GS.N52.F4; both cell clones are derived from the original cell line GS.N52. 20 6. Characterization of the El cell lines a) Southern blot analyses Southern blot analyses were carried out in order to investigate the integration status of the El 25 region in the cell lines. This was done by isolating genomic DNA from all eight El amniotic clones (see Section 5.), and 5 pg of each were digested with EcoRV, fractionated by electrophoresis and transferred to a nylon membrane. EcoRV cuts once in the El expression 30 cassette. Hybridization with radiolabeled STK146 DNA confirmed integration of 1-2 copies of STK146 in all clones. Fig. 3 shows the integration pattern in the El cell clones. There are mainly two high molecular weight bands evident for all the clones, which indicates 35 integration of a single copy. The cell clones GS.N24 and GS.N52 each showed an additional band with relatively strong intensity, which might indicate integration of tandem copies of STK146. For none of the - 48 cell clones were there any bands smaller than STK146 digested with ScaI and EcoRV, which suggested that all the integrates were present completely and not deleted. 5 b) Generation of recombinant adenoviruses After the single-cell cloning, the cell clones were tested for their ability to generate recombinant adenovirus. This was done once by infecting 3-5 single cell clones of each cell line in approx. 70% confluent 10 24-well plates with about 5 moi (multiplicity of infection) of Adpgal (recombinant first-generation adenoviral vector) . 48 hours after the infection, the cells were lysed by freezing and thawing three times, and the amount of Adpgal produced was analyzed by 15 infecting 293 cells and subsequently staining the cells (MacGregor *et al., in: Gene Transfer and. Expression Protocols, Murray ed. Humana, Clifton, NJ, vol. 7, pp. 217-235, year (1991)) for detecting p-galactosidase production (Fig. 4). This method affords an only 20 approximate production of recombinant adenovirus because the number of cells and therefore the amount of virus used may differ in the different cell clones because of their size and growth rate. The cell clones which gave the largest yield in this first test were 25 analyzed more exactly in a further experiment. This was done by seeding about 3 x 107 cells on 3 dishes (diameter 100 mm, surface area 60 cm 2 ) . The cells were counted the next day and were infected with exactly 5 moi of Adpgal based on the number of cells found. 30 48 hours after the infection, the cells were harvested and lysed by freezing and thawing three times, and the amount of Adpgal produced was analyzed by infecting 293 cells and subsequently staining. The result is depicted in Fig. 4. 35 c) Transfection efficiency Some of the cloned cell lines were tested for plaque formation. This was done by transfecting approx.

- 49 70% confluent cell culture dishes (diameter 60 mm) with 2 pg of infectious plasmid GS66 by the calcium phosphate method. Plasmid GS66 contains the complete adenovirus genome with a deletion in the El region from 5 nucleotide 440 to nucleotide 3523. The adenoviral terminal repeat sequences (ITRs) are flanked in this plasmid by SwaI restriction cleavage sites, so that infectious virus and plaques can be produced after transfection of SwaI-digested plasmid. About 24 hours 10 after transfection, the cells were covered with about 10 ml of MEM, 1% agarose, 0.5x penicillin/strepromycin, 0.05% yeast extract. Plaques were visible after incubation at 37 0 C, 95% humidity, 5% CO 2 for about 1 week. Fig. 4 shows the number of counted plaques 15 averaged for 2 independent transfections in each case. d) Expression of ElA and E1B functions of Ad5 Expression of the Ad5-E1A and of the E1B-21kD proteins in the cloned cell lines was detected by 20 Western blot analyses using monoclonal antibodies. The cells were carefully detached from a cell culture (diameter 10 cm) in PBS/1mM EDTA, pelleted and taken up in 150 tl of 50 mM tris/HCL PH 8, 140 mM NaCl, 0.5% NP40, 4 mM EDTA, 2 mM EGTA, 0.5 mM PMSF, 5% 25 glycerol. The cells were lyzed by additional Dounce grinding and centrifuged down at 13,000 rpm for 10 minutes, and the protein concentration in the supernatant was determined using a protein determination kit (BIORAD, Microassay Procedure). 10 ptg 30 of protein were fractionated on a 12% SDS polyacrylamide gel, transferred to a nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech) and incubated with an anti-ElA or anti-ElB 21kD antibody (Calbiochem, dilution 1:300) . The next day, the blot 35 was washed and hybridized with a second anti-mouse antibody (ElA) or anti-rat antibody (ElB) to which horseradish peroxidase was coupled. The horseradish peroxidase reaction was started by incubation of equal - 50 volumes of enhance solution 1 and 2 (ECL, Amersham Pharmacia Biotech), and the photochemical reaction which developed thereby was visualized by brief exposure to an X-ray film. Fig. 5 shows the result of a 5 Western blot analysis. e) Time-course of the synthesis of recombinant first generation adenoviral vectors and adenoviral vectors with large DNA capacity 10 Two cloned cell lines N52.E6 and N52.F4 showed the highest yield of recombinant first-generation adenoviral vectors in the experiments described above. Further knowledge about the time course is important for optimal production of adenoviral vectors, 15 especially when these cells are adapted to suspension cultures and the success of infection cannot be followed by means of a cytopathic effect. For this analysis, several dishes (diameter 6 cm) each with 3 x 106 cells were infected with 5 moi of Adpgal and 20 harvested at the stated times after the infection. The yield of recombinant adenovirus was again determined by infecting 293 cells, staining and counting the blue cells (Fig. 6 A). It is intended in future also to use the new 25 cell lines for producing adenoviral vectors with large DNA capacity (see above). Production of these adenoviral vectors requires helper viruses which supply the deleted functions and proteins for a lytic infection cycle in trans. The packaging signal of these 30 helper viruses is deleted with the aid of loxP recognition sequences and Cre recombinase, which is expressed by the cell line, on infection. It is therefore intended in future experiments that the new El-transformed cell lines will be transfected with a 35 Cre-expressing plasmid, and the recombinase will be expressed stably. It has been tested in preliminary experiments whether the new El amniocytes are also able to produce - 51 adenoviral vectors of large DNA capacity, and whether production kinetics and the amount of produced vectors corresponds to that achieved with the existing Cre expressing 293 cells. For this purpose, several dishes 5 each with 3 x 106 cells of the cell lines N52.E6 and N52.F4 were infected with 5 MOI of loxP helper virus and 10 MOI of AdGS46 (P-gal-expressing adenoviral vector with large DNA capacity) and harvested at the stated times after the infection. The yield of p-gal 10 expressing adenoviral vector with large DNA capacity was again determined by infecting 293 cells, staining and counting the blue cells (Fig. 6B) . The amount of adenoviral vectors of large DNA capacity synthesized in the amniocytes corresponds to that also produced in 15 Cre-expressing 293 cells (data not shown).

Claims (21)

1. A permanent amniocytic cell line comprising at least one nucleic acid which brings about expression of the gene products of the adenovirus ElA and E1B 5 regions.

2. A cell line as claimed in claim 1, where the at least one nucleic acid also brings about expression of the gene products of the adenovirus E2A, E2B and/or E4 regions and/or of Cre recombinase. 10

3. A cell line as claimed in claim 1 or 2, where expression of the gene product of the ElA region is under the control of a constitutive promoter, preferably the phosphoglycerate kinase (PGK) promoter.

4. A cell line as claimed in any of claims 1-3, 15 where expression of the gene product(s) of the E1B region is under the control of an adenoviral promoter, preferably the adenoviral ElB promoter.

5. A cell line as claimed in any of claims 1-4, where the adenoviral gene products are derived from 20 human adenovirus type 5.

6. A cell line as claimed in any of claims 1-5, where the cell line is a human cell line.

7. A process for producing a permanent amniocytic cell line which comprises the transfection of 25 amniocytes with at least one nucleic acid which brings about expression of the adenoviral gene products of the ElA region and E1B region.

8. A process as claimed in claim 7, where primary amniocytes, in particular human primary amniocytes, are 30 used.

9. A process as claimed in claim 7 or 8, where said nucleic acid is used in the form of an expression vector.

10. A process as claimed in any of claims 7 - 9, 35 where expression of the gene product of the ElA region is under the control of a constitutive promoter, preferably the phosphoglycerate kinase (PGK) promoter, and of the gene product(s) of the ElB region is under - 53 the control of an adenoviral promoter, preferably the adenoviral ElB promoter.

11. A process as claimed in any of claims 7-10, where transfection of the amniocytes and/or of the 5 resulting cell line additionally brings about expression of the gene products of the adenovirus E2A and/or E2B and/or E4 regions and/or of Cre recombinase.

12. A process as claimed in any of claims 7 - 11 where the adenoviral gene products are derived from 10 human adenovirus type 5.

13. A permanent amniocytic cell line obtainable by the process as claimed in any of claims 7 - 12.

14. The permanent amniocytic cell line N52.E6 (DSM ACC2416).

15 15. The use of amniocytes for producing adenovirus transformed permanent amniocytic cell lines.

16. The use of the adenoviral gene products of the ElA and ElB regions for producing permanent amniocytic cell lines. 20

17. The use of a permanent amniocytic cell line for producing gene transfer vectors and/or adenovirus mutants.

18. The use as claimed in claim 17 for producing adenovirus vectors, AAV (adeno-associated virus) 25 vectors, retrovirus vectors, lentivirus vectors, chimeric adenovirus-AAV vectors, chimeric adenovirus AAV vectors [sic], chimeric adenovirus-retrovirus vectors and/or chimeric adenovirus-lentivirus vectors.

19. The use as claimed in claim 18, where the 30 adenovirus vectors are first-generation adenovirus vectors, second-generation adenovirus vectors, adenovirus vectors of large DNA capacity and/or deleted adenovirus vectors.

20. The use as claimed in any of claims 17-19 for 35 producing tropism-modified gene transfer vectors and/or tropism-modified adenovirus mutants. - 54

21. The use as claimed in any of claims 17-20, where the amniocytic cell line is as defined in any of Claims 1-6, 13 or 14.