NON SELF-INACTIVATING, EXPRESSION TARGETED RETROVIRAL VECTORS

Abstract: The present invention relates to a retroviral vector undergoing promoter conversion comprising a 5'LTR region of the structure U3-R-U5; one or more sequences selected from coding and non-coding sequences; and a 3'LTR region comprising a completely or partially deleted U3 region wherein said deleted U3 region is replaced by a polylinker sequence, followed by the R and U5 region. Said retroviral vector undergoes promoter conversion and is useful as a gene transfer vehicle for targeted gene therapy.

Description: NON SELFJNACTIVATING, EXPRESSION TARGETED RETROVIRAL VECTORS The present invention relates to retroviral vectors including a vector which undergoes promoter conversion (ProCon vector). The vector system is useful as a gene transfer vehicle for targeted gene therapy. Background of the Invention The use of retroviral vectors for gene therapy has received much attention and currently is the method of choice for the transferral of therapeutic genes in a variety of approved protocols both in the USA and in Europe (Kotani et al.. 1994). However most of these protocols require that the infection of target cells with the retroviral vector carrying the therapeutic gene occurs in vitro, and successfully infected cells are then returned to the affected individual (Rosenberg et al.. 1992; for a review see Anderson, 1992). Such ex vivo gene therapy protocols are ideal for correction of medical conditions in which the target cell population can be easily isolated (e.g. lymphocytes). Additionally the ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use. Unfortunately, only a fraction of the possible applications for gene therapy involve target cells that can be easily isolated, cultured and then reintroduced. Additionally, the complex technology and associated high costs of ex vivo gene therapy effectively preclude its disseminated use world-wide. Future facile and cost-effective gene therapy will require an in vivo approach in which the viral vector, or cells producing the viral vector, are directly administered to the patient in the form of an injection or simple implantation of retroviral vector producing cells. This kind of in vivo approach, of course, introduces a variety of new problems. First of ail, and above all, safety considerations have to be addressed. Virus will be produced, possibly from an implantation of virus producing cells, and there will be no opportunity to precheck the produced virus. It is important to be aware of the finite risk involved in the use of such systems, as well as trying to produce new systems that minimize this risk. Retroviral vector systems consist of two components (Fig. 1): 1) the retroviral vector itself is a modified retrovirus (vector plasmid) in which the genes encoding for the viral proteins have been replaced by therapeutic genes and marker genes to be transferred to the target cell. Since the replacement of the genes encoding for the viral proteins effectively cripples the virus it must be rescued by the second component in the system which provides the missing viral proteins to the modified retrovirus. The second component is: 2) a cell line that produces large quantities of the viral proteins, however lacks the ability to produce replication competent virus. This cell line is known as the packaging cell line and consists of a cell line transfected with a second plasmid carrying the genes enabling the modified retroviral vector to be packaged. To generate the packaged vector, the vector plasmid is transfected into the packaging cell line. Under these conditions the modified retroviral genome including the inserted therapeutic and marker genes is transcribed from the vector plasmid and packaged into the modified retroviral particles (recombinant viral particles). This recombinant virus is then used to infect target cells in which the vector genome and any carried marker or therapeutic genes becomes integrated into the target cell's DNA. A cell infected with such a recombinant viral particle cannot produce new vector virus since no viral proteins are present in these cells. However the DNA of the vector carrying the therapeutic and marker genes is integrated in the cell's DNA and can now be expressed in the infected cell. The essentially random integration of the proviral form of the retroviral genome into the genome of the infected cell (Varmus, 1988) led to the identification of a number of cellular proto-oncogenes by virtue of their insertional activation (Varmus, 1988; van Lohuizen and Berns, 1990). The possibility that a similar mechanism may cause cancers in patients treated with retroviral vectors carrying therapeutic genes intended to treat other pre-existent medical conditions has posed a recurring ethical problem. Most researchers would agree that the probability of a replication defective retroviral vector, such as all those currently used, integrating into or near a cellular gene involving in controlling cell proliferation is vanishingly small. However it is generally also assumed that the explosive expansion of a population of replication competent retrovirus from a single infection event, will eventually provide enough integration events to make such a phenotypic integration a very real possibility. Retroviral vector systems are optimized to minimize the chance of replication competent virus being present. However it has been well documented that recombination events between components of the retroviral vector system can lead to the generation of potentially pathogenic replication competent virus and a number of generations of vector systems have been constructed to minimize this risk of recombination (reviewed in Salmons and Gϋnzburg, 1993). A further consideration when considering the use of in vivo gene therapy, both from a safety stand point and from a purely practical stand point, is the targeting of retroviral vectors. It is clear that therapeutic genes carried by vectors should not be indiscriminately expressed in all tissues and cells, but rather only in the requisite target cell. This is especially important if the genes to be transferred are toxin genes aimed at ablating specific tumour cells. Ablation of other, nontarget cells would obviously be very undesirable. A number of retroviral vector systems have been previously described that should allow targeting of the carried therapeutic genes (Salmons and Gunzburg, 1993). Most of these approaches involve either limiting the infection event to predefined cell types or using heterologous promoters to direct expression of linked heterologous therapeutic or marker genes to specific cell types. Heterologous promoters are used which should drive expression of linked genes only in the cell type in which this promoter is normally active. These promoters have previously been inserted, in combination with the marker or therapeutic gene, in the body of the retroviral vectors, in place of the gag, pol or env genes. The retroviral Long Terminal Repeat (LTR) flanking these genes carries the retroviral promoter, which is generally non-specific in that it can drive expression in many different cell types (Majors, 1990). Promoter interference between the LTR promoter, and heterologous internal promoters, such as the tissue specific promoters described above has been reported. Additionally, it is known that retroviral LTRs harbor strong enhancers that can, either independently, or in conjunction with the retroviral promoter, influence expression of cellular genes near the site of integration of the retrovirus. This mechanism has been shown to contribute to tumorigenicity in animals (van Lohuizen and Bems). These two observations have encouraged the development of Self-lnactivating-Vectors (SIN) in which retroviral promoters are functionally inactivated in the target cell (PCT WO94/29437). Further modifications of these vectors include the insertion of promoter gene cassettes within the LTR region to create double copy vectors (PCT. WO 89/11539). However, in both these vectors the heterologous promoters inserted either in the body of the vector, or in the LTR region are directly linked to the marker/therapeutic gene. The previously described SIN vector mentioned above carrying a deleted 3'LTR (PCT WO9429437) utilize in addition a strong heterologous promoter such as that of Cytomegalovirus (CMV), instead of the retroviral 5'LTR promoter (U3-free 5 'LTR) to drive expression of the vector construct in the packaging cell line. A heterologous polyadenylation signal is also included in the 3 'LTR (PCT W09429437). The object of the present invention is the construction of a novel retroviral vector which can be used as a safe gene transfer vehicle for targeted gene therapy with a reduced probability to undergo recombination with the packaging construct. This novel vector carries heterologous promoter and/or regulatory elements in the 3 'LTR which, after infection become duplicated and translocated to the 5 ' LTR in the target cell, eventually controlling expression of marker/therapeutic genes, not directly linked to the promoter, but rather inserted into the body of the vector. This vector does not undergo self-inactivation - but instead promoter exchange, giving rise to the name ProCon for Promoter Conversion. Since Promoter Conversion does not result in Self-lnactivation, the retroviral vector will be transcriptionally active in the target cell. However both LTRs will consist to a large extent of heterologous promoter/enhancer sequences in the target cell. This will reduce the likelihood of the integrated vector in the target cell being subject to the same inactivation over long periods as has been described for conventional vectors (Xu et al., 1989) and also will reduce the chance of recombination with endogenous retroviral sequences to generate potentially pathogenic replication competent virus, increasing the safety of the system. In this invention the 5 'LTR of the retroviral vector construct is not modified, and expression of the viral vector in the packaging cells is driven by the normal retroviral U3 promoter. Normal retroviral polyadenylation is allowed, and no heterologous polyadenylation signals are included in the 3 ' LTR. This is important for the development of in vivo gene therapy strategies, since the normal physiological regulation of the virus, through the normal viral promoter, and possibly also involving the normal viral control of poiyadenylation, will prevail over long periods in vivo whilst the packaging cells are producing recombinant virus. A further modification of this novel retroviral vector forsees the inclusion of cellular sequences instead of the heterologous promoter and/or regulatory elements. This should allow higher selectivity for site specific recombinantion with cellular sequences to target the integration of retroviral vectors to particular sites in the host cell genome (Sailer, 1994). To achieve the foregoing and other objects, the invention provides a retroviral vector undergoing promoter conversion comprising a 5' LTR region of the structure U3-R- U5; one or more sequences selected from coding and non-coding sequences; and a 3' LTR region comprising a completely or partially deleted U3 region wherein said deleted U3 region is replaced by a polylinker sequence, followed by the R and U5 region. Said polylinker sequence carries at least one unique restriction site and contains preferably at least one insertion of a heterologous DNA fragment. Said heterologous DNA fragment is preferably selected of regulatory elements and promoters, preferably being target cell specific in their expression, but may also be a DNA fragment with no regulatory function. Said heterologous DNA fragment is preferably homologous to one or more cellular sequences. The regulatory elements and promoters are preferably regulatable by transacting molecules. Further objects, features and advantages will be apparent from the following description of preferred embodiments of the invention. The target cell specific regulatory elements and promoters are selected from one or more elements of the group consisting of Whey Acidic Protein (WAP), Mouse Mammary Tumour Virus (MMTV), β-lactoglobulin and casein specific regulatory elements and promoters, pancreas specific regulatory elements and promoters including carbonic anhydrase II and β-glucokinase regulatory elements and promoters, lymphocyte specific regulatory elements and promoters including immunogiobulin and MMTV lymphocytic specific regulatory elements and promoters and MMTV specific regulatory elements and promoters conferring responsiveness to glucocorticoid hormones or directing expression to the mammary gland. Said regulatory elements and promoters regulate preferably the expression of at least one of the coding sequences of said retroviral vector. The LTR regions are selected from at least one element of the group consisting of LTRs of Murine Leukaemia Virus (MLV), Mouse Mammary Tumour Virus (MMTV), Murine Sarcoma Virus (MSV), Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus (HIV), Human T-ceil Leukaemia Virus (HTLV), Feline Immunodeficiency Virus (FIV), Feline Leukaemia Virus (FELV), Bovine Leukaemia Virus (BLV) and Mason-Pfizer-Monkey virus (MPMV). The retroviral vector is based preferably either on a BAG vector (Price etal., 1987) or an LXSN vector (Miller and Rosman, 1989). The coding sequence is preferably selected from one or more elements of the group consisting of marker genes, therapeutic genes, antiviral genes, antitumour genes, cytokine genes. Said marker and therapeutic genes are preferably selected from one or more elements of the group consisting of β-galactosidase gene, neomycin gene, Herpes Simplex Virus thymidine kinase gene, puromycin gene, cytosine deaminase gene, hygromycin gene, secreted alkaline phosphatase gene, guanine phosphoribosyl transferase (gpt) gene, alcohol dehydrogenase gene and hypoxanthine phosphoribosyl transferase (HPRT) gene. Another embodiment of the invention envisages the alteration or partial deletion of at least one retroviral sequence required for integration of retroviruses. In a further embodiment of the invention a retroviral vector system is provided comprising a retroviral vector as described above as a first component and a packaging cell line harbouring at least one retroviral or recombinant retroviral construct coding for proteins required for said retroviral vector to be packaged. The packaging cell line harbours retroviral or recombinant retroviral constructs coding for those retroviral proteins which are not encoded in said retroviral vector. The packaging cell line is preferably selected from an element of the group consisting of psi-2, psi-Crypt, psi-AM, GP+E-86, PA317 and GP+envAM-12, or of any of these supertransfected with recombinant constructs allowing expression of surface proteins from other enveloped viruses. Another embodiment of the invention involves the use of a packaging cell line harbouring a recombinant retroviral construct defective in integrase function. After introducing the retroviral vector of the invention as described above in a retroviral packaging cell line and infection of a target cell, as described above, a retroviral provirus is provided wherein said polylinker and any sequences inserted in said polylinker in the 3'LTR become duplicated during the process of reverse transcription in the infected target cell and appear in the 5'LTR as well as in the 3'LTR of the resulting provirus. The invention includes also mRNA of a retroviral provirus according to the invention and any RNA resulting from a retroviral vector according to the invention. A further embodiment of the invention provides non-therapeutical method for introducing homologous and/or heterologous nucleotide sequences into human or animal cells in vitro and in vivo comprising transfecting a packaging cell line of a retroviral vector system according to the invention with a retroviral vector according to the invention and infecting a target cell population with recombinant retroviruses produced by the packaging cell line. The nucleotide sequences are selected from one or more elements of the group consisting of genes or parts of genes encoding for proteins, regulatory sequences and promoters. The retroviral vector, the retroviral vector system and the retroviral provirus as well as RNA thereof is used for producing a pharmaceutical composition for gene therapy in mammals including humans. Furthermore, they are used for targeted integration in homologous cellular sequences. Promoter conversion The present invention uses the principle of promoter conversion typical for retroviruses. The retroviral genome consists of an RNA molecule with the structure R-U5-gag-pol- env-U3-R (Fig. 2). During the process of reverse transcription, the U5 region is duplicated and placed at the right hand end of the generated DNA molecule, whilst the U3 region is duplicated and placed at the left hand end of the generated DNA molecule (Fig. 2). The resulting structure U3-R-U5 is called LTR (Long Terminal Repeat) and is thus identical and repeated at both ends of the DNA structure or provirus (Varmus, 1988). The U3 region at the left hand end of the provirus harbours the promoter (see below). This promoter drives the synthesis of an RNA transcript initiating at the boundary between the left hand U3 and R regions and terminating at the boundary between the right hand R and U5 region (Fig. 2). This RNA is packaged into retroviral particles and transported into the target cell to be infected. In the target cell the RNA genome is again reverse transcribed as described above. According to the invention a retroviral vector is constructed in which the right- hand U3 region is altered (Fig. 3), but the normal left-hand U3 structure is maintained (Fig. 3); the vector can be normally transcribed into RNA utilizing the normal retroviral promoter located within the left-hand U3 region (Fig. 3). However the generated RNA will only contain the altered right-hand U3 structure. In the infected target cell, after reverse transcription, this altered U3 structure will be placed at both ends of the retroviral structure (Fig. 3). If the altered region carries a polylinker (see below) instead of the U3 region then any promoter, including those directing tissue specific expression such as the WAP promoter (see below) can be easily inserted. This promoter will then be utilized exclusively in the target cell for expression of linked genes carried by the retroviral vector. Altematively or additionally DNA segments homologous to one or more celluar sequences can be inserted into the polylinker for the purposes of gene targeting, by homologous recombination(see below). According to the invention the term "polylinker" is used for a short stretch of artificially synthesized DNA which carries a number of unique restriction sites allowing the easy insertion of any promoter or DNA segment. The term "heterologous" is used for any combination of DNA sequences that is not normally found intimately associated in nature. Gene expression is regulated by promoters. In the absence of promoter function a gene will not be expressed. The normal MLV retroviral promoter is fairly unselective in that it is active in most cell types (Majors, 1990). However a number of promoters exist that show activity only in very specific cell types. Such tissue-specific promoters will be the ideal candidates for the regulation of gene expression in retroviral vectors, limiting expression of the therapeutic genes to specific target cells. In the packaging cell line the expression of the retroviral vector is regulated by the normal unselective retroviral promoter contained in the U3 region (Fig. 3). However as soon as the vector enters the target cell promoter conversion occurs, and the therapeutic or marker gene, e.g. b-galactosidase are expressed from a tissue specific promoter of choice introduced into the polylinker (Fig. 3). Not only can virtually any tissue specific promoter be included in the system, providing for the selective targeting of a wide variety of different cell types, but additionally, following the conversion event, the structure and properties of the retroviral vector no longer resembles that of a virus. This, of course, has extremely important consequences from a safety point of view, since ordinary or state of the art retroviral vectors readily undergo genetic recombination with the retroviral packaging construct and/or endogenous retroviruses to produce potentially pathogenic viruses. Promoter conversion (ProCon) vectors do not resemble retroviruses because they no longer carry U3 retroviral promoters after conversion thus reducing the possibility of genetic recombination. The retroviral promoter structure is carried within the U3 region of the LTR. LTRs carry signals that allow them to integrate into the genome of the target cell. The integration of retroviral proviruses can also contribute to pathogenic changes (van Lohuizen and Bems, 1990). In one embodiment of the invention ProCon vectors can carry modified LTRs that no longer carry the signals required for integration. Again this increases the potential safety of these vector systems. Gene Targeting According to another aspect of the present invention the retroviral vector is used for targeted integration into the target cell. The integration of the proviral DNA version of the retroviral genome into the target cell is a major advance to the use of retroviruses as vectors when compared to other viruses such as adenoviruses, since it allows long term stable expression of transferred genes. However the random nature of this integration event also poses a major disadvantage to the use of retroviral vectors since it raises the possibility of insertional (in)activation of cellular tumour suppressor genes or proto-oncogenes and thus tumour induction (van Lohuizen and Berns, 1990). Homologous recombination has been successfully used to target the integration of transfected or microinjected DNA to specific DNA loci and is routinely used in the construction of "knock-out" transgenic mice or animals (reviewed in Capecchi, 1989; Bradley etal., 1992; Morrow and Kucherlapati, 1993). Unfortunately the efficiency of DNA transfer by such purely physical methods is extremely low. In contrast retroviral mediated gene transfer is very efficient, almost 100% of a population of cells being infectable. A combination of retroviral gene transfer with homologous recombination should allow the construction of an ideal system for locus targeted integration. We have investigated the feasibility of introducing long homologous pieces of DNA into retroviral vectors in different locations to promote integration by homologous recombination (Sailer, 1994). Both gene conversion and homologous recombination have been evaluated. Using a cell line carrying a single copy of the HSV-tk gene as a target we have been able to disrupt the target at frequencies 15 fold higher than previously reported by others (Ellis and Bernstein, 1989). Cloning of the recombined fragments of DNA has revealed the presence of both target tk sequence and retroviral vector (Sailer, 1994). For targeted integration DNA segments homologous to cellular sequences are inserted into the polylinker of the ProCon vectors. After infection of the target cell and reverse transcription, these sequences will appear at the 5' terminal end of the provirus. Terminal homologies have been shown to favour homologous recombination (Bradley, 1991) to isogenic cellular sequences (Bradley, 1991). Infection of target cells which carry mutated versions of the homologous sequence should result in the recombination and thus repair of the mutated sequence. Either just the homologous sequences will recombine into the cellular genome, or the complete vector will be inserted (Saller,1994). Not only has this vector class potential for use in gene repair, it can also be utilized to direct the integration of retroviral vectors carrying therapeutic genes to specific loci in the genome which are known not to harbour active genes. This will reduce considerably the possibility of insertional activation or inactivation as described above, and will thus contribute to the safety of the use of retroviral vectors. The following examples will illustrate the invention further. These examples are however in no way intended to limit the scope of the present invention as obvious modifications will be apparent, and still other modifications and substitutions will be apparent to anyone skilled in the art. The recombinant DNA methods employed in practicing the present invention are standard procedures, well known to those skilled in the art, and described in detail, for example, in "Molecular Cloning" (Sambrook et al. 1989) and in "A Practical Guide to Molecular Cloning" (Perbal, 1984). Brief description of the drawings refered to in the following examples: Fig. 1 ; Retroviral vector system Fig. 2 Retroviral genome, reverse transcription Fig. 3 ProCon principle Fig. 4 PCR analysis, MLV probe Fig. 5 PCR analysis, MMTV probe Fig. 6 β-galactosidase expression in infected NIH and EF43 cells Fig. 7: β-galactosidase expression in infected primary mammary glands cells from a pregnant mouse Fig. 8: β-galactosidase expression after virus injection into mammary gland and the skin of a pregnant Balb/c mouse Fig. 9: β-galactosidase expression in infected mammary tumour cells Fig. 10: Targeted integration of a retroviral vector by homologous recombination Example 1 Mammary gland specific expression after infection with ProCon Vectors carrying mammary specific promoters. In the murine leukemia virus (MLV) retroviral vector known as BAG (Price et al., 1987) the β-galactosidase gene is driven by the promiscuous (i.e. non-tissue specific) MLV promoter in the U3 region of the LTR (Fig. 3). According to the present invention a derivative of the BAG vector has been constructed in which the MLV promoter (U3) located within the 3'LTR (Fig. 3) has been deleted and replaced with a polylinker, said polylinker allowing the facile introduction of heterologous promoters. The BAG vector lacking the U3 is expressed from the MLV promoter (U3) within the 5'LTR when introduced into a packaging cell line. As a result of the rearrangements occurring in the retroviral genome during its life cycle, following infection of its target cell, the polylinker will be duplicated at both ends of the retroviral genome as described above. Thereby a retroviral vector can be constructed in which the expression of the β-galactosidase gene of BAG will be controlled by any heterologous promoter inserted into the polylinker in the target cell (Fig. 3). According to the principle set forth above the following specific promoters have been inserted into the polylinker region or the modified BAG vector: Several subregions of the Mouse Mammary Tumour Virus (MMTV) promoter including a region that confers responsiveness to glucocorticoid hormones and a region containing an element that directs expression to the mammary gland. The Whey Acidic Protein (WAP) promoter. This promoter controls the expression of WAP so that it is only produced in the mammary glands of pregnant and lactating rodents. The control of the β-galactosidase gene expression by promoters inserted into the polylinker has been validated by infection studies using the constructed MMTV and WAP retroviral vectors to infect various cells. To produce retroviral vector particles, the MMTV and WAP ProCon vectors have been transfected into the packaging cell line GP+E86 (Markowitz et al., 1988). After selection for neomycin resistance, which is encoded by the vector, stable populations and clones of recombinant ProCon virus producing cells were obtained. Virus containing supematant from these populations was used to infect a mouse mammary cell line EF43 (Gϋnzburg et al.. 1988) as well as a mouse fibroblast cell line (Jainchill et al.. 1969). Four days after infection the target cells were lysed and quantitative β- galactosidase assay revealed no expression in either cell type infected by the WAP carrying ProCon vectors and good expression in both cell types from the MMTV carrying ProCon vector (Fig. 6). This result is in accordance with the WAP promoter only functioning in vivo during late pregnancy and lactation and not in most simple ID vitro mammary cell culture systems as represented by the EF43 cells. To investigate whether the WAP carrying ProCon vectors would be active in a complex primary mammary derived cell culture system, primary organoids from 8-10 day pregnant mice (Fig. 7) or from mammary tumours (Fig. 9) were taken into culture and infected with the supematant from the same stably transfected population of transfected cell lines. Both ProCon vectors carrying the WAP and the MMTV promoter fragments were active in these primary cells (Fig. 7) and mammary tumour derived cells (Fig. 9) as demonstrated by β-galactosidase activity. To investigate whether the WAP and MMTV carrying ProCon vectors were active jn vivo and whether the expression of β-galactosidase was limited to the mammary gland in vivo, recombinant ProCon virus containing medium was injected in situ into the mammary glands or skin of 8-10 day pregnant mice. Five days later the mice were sacrificed, cell extracts prepared and a β-gaiactosidase assay performed. Both the WAP and MMTV fragment carrying ProCon vectors were expressed only in the pregnant mammary gland and not in the skin (cf M and S in Fig. 8). Thus in vivo the regulatory elements from both promoters limit expression to the mammary gland whereas in vitro the regulatory elements from the WAP promoter retain their strict tissue specificity but those of MMTV do not. These ProCon vectors carrying tissues specific promoters and regulatory elements will be useful for directing the expression of therapeutic genes to predefined cell types, tissues and organs. Potential therapeutic genes include melittin, which has anti-HIV and anti-tumour effects, and genes which prime cells for death including the thymidine kinase, guanine phosphoribosytransferase and cytosine deaminase genes, cytochrome P450 as well as genes involved in cell cycle regulation such as SDI WAF- 1/CIP-1. Example 2 Validation of promoter conversion in cells infected with a ProCon vector that originally carried the MMTV promoter in the 3'LTR. A ProCon vector carrying the promoter region from mouse mammary tumour virus (MMTV) was transfected into a packaging cell line and the resultant recombinant vector particles used to infect an established human bladder carcinoma cell line (EJ). Infected cell clones were selected in medium carrying the neomycin analog G418 (since the vector carries a neomycin resistance gene driven from an internal SV40 promoter). DNA was prepared from one of the infected clones and nontransfected parental EJ cells and used for Polymerase Chain Reactions (PCR). The PCRs were performed using one of two primers that specifically recognise and bind to MMTV sequences (A, B in Figs. 4 & 5) or the MLV R region (C in Fig. 4) of the LTR together with a primer located within the marker gene (Figs. 4 & 5). Since the marker gene primer is only located down stream of the MMTV (or MLV R region) sequence if promoter conversion has occurred, a positive PCR signal obtained with the MMTV primers in combination with the marker gene primer is indicative of this. In Fig. 4 the PCR products using primers A, B or C are shown after hybridization to a labelled fragment from the MLV sequence, verifying that all three PCR products are of MLV origin. The size of the fragments verifies that promoter conversion has occured. Fig. 5 shows the PCR products using primer A or B and hybdridized to an MMTV specific probe, again verifying that promoter conversion has occured. Example 3 Const ction of ProCon Vectors for targeted integration. Using the same BAG vector described in Example 1 above, a retroviral vector can be constmcted in which a DNA sequence with homology to a cellular sequence can be inserted into the LTR. The resulting vector can be used to target the integration of either the homologous sequence inserted into the vector or the whole or part of the vectors into the homologous sequence present in the host cell genome. According to the principle set forth above, a fragment of the thymidine kinase (tk) gene of heφes simplex vims (HSV) has been inserted into the polylinker region of the modified BAG vector (tk mutant in Fig. 10, Sailer, 1994). A cell line has also been established that has no functional copy of the mammalian tk gene and instead carries one copy of the HSV-tk gene (Sailer, 1994). This cell line has been infected with the tk carrying BAG vector and cells that have undergone disruption of the HSV-tk gene have been selected (Fig. 10). The above examples have illustrated the principles and consequences of the promoter conversion vectors provided by the present invention. References Anderson, W.F. 1992. Human gene therapy. Science 256: 808-813. Bradley, A. 1991. Modifying the mammalian genome by gene targeting. Curr. Opin. Biotechnol. 2: 823-829. Bradley, A., P. Hasty, A. Davis, and R. Ramirez-Solis. 1992. Modifying the mouse: design and desire. Bio/technology 10: 534-539. Capecchi, M.R. 1989. Altering the genome by homologous recombination. Science 244: 1288-1292. Ellis, J. and A. Bernstein. 1989. Gene Targeting with Retroviral Vectors Recombination by Gene Conversion into Regions of Nonhomology. Mol. Cell. Biol. 9: 1621 -1627. Emerman, M. and H.M. Temin. 1986. Comparison of promoter suppression in avian and murine retrovirus vectors. Nucl. Acids Res. 14: 9381-9396. Gϋnzburg, W.H., B. Salmons, A. Schlaeffli, S. Moritz-Legrand, W. Jones, N.H. Sarkar, and R. Ullrich. 1988. Expression of the oncogenes mil and ras abolishes the in vivo differentiation of mammary epithelial cells. Carcinogenesis 9: 1849-1856. Jainchill, J.L., S.A. Anderson, and G.J. Todaro. 1969. Murine sarcoma and leukaemia vi ses: assay using clonal lines of contact-inhibited mouse cells. J. Virol 4: 549-553. Kotani, H., P.B. Newton, S. Zhang, Y.L. Chiang, E. Otto, L. Weaver, R.M. Blaese, W.F. Anderson, and G.J. McGarrity. 1994. Improved methods of retroviral vector transduction and production for gene therapy. Human Gene Therapy 5: 19-28. Majors, J. 1990. The structure and function of retroviral long terminal repeats, Curr. Tops. In Micro. Immunol. 157: 49-92. Markowitz, D., S. Goff, and A. Bank. 1988. A safe packaging line for gene transfer separating viral genes on two different plasmids. J. Virol. 62: 1120-1124. Miller, A.D. and G.J. Rossman. 1989. Improved retroviral vectors for gene transfer and expression. Biotechniques 7: 980-990. Morrow, B. and R. Kucheriapati. 1993. Gene targeting in mammalian cells by homologous recombination. Current Opinion in Biotechnology 4: 577-582. Perbal, B. 1984. A Practical Guide to Molecular Cloning, John Wiley & Sons. Price, J., D. Turner, and C. Cepko. 1987. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc. Natl. Acad. Sci. USA 84: 156-160. Rosenberg, S.A., Anderson, W.F., Blaese, R.M. et al. 1992. Immunization of cancer patients using autologous cancer cells modified by insertion of the gene for interleukin-2. Hum. Gene Ther. 3: 75-90. Sailer, R.M. 1994 Design von locus- und gewebespezischen retroviralen Vektoren fuer eine in vivo Gentherapie. Doctoral thesis, Ludwigs-Maximilians University Munich, Germany. Salmons, B. and W.H. Gϋnzburg. 1993. Targeting of retroviral vectors for gene therapy. Human Gene Therapy 4: 129-141. Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular Cloning. Cold Spring Harbor Laboratory Press, New York, USA van Lohuizen and Bems. 1990. Tumorigenesis by slow-transforming retroviruses - an update. Biochim. Biophys. Acta, 1032: 213-235. Varmus, H. 1988. Retrovimses. Science 240: 1427-1435. Xu, L, J.-K. Yee, J.A. Wolff and T. 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Claims: C L A I M S 1. A retroviral vector undergoing promoter conversion comprising a 5' LTR region of the stmcture U3-R-U5; one or more sequences selected from coding and non- coding sequences; and a 3' LTR region comprising a completely or partially deleted U3 region wherein said deleted U3 region is replaced by a polylinker sequence, followed by the R and U5 region. 2. A retroviral vector according to claim 1 comprising a 5' LTR region of the structure U3-R-U5; one or more sequences selected from coding and non-coding sequences; and a 3' LTR region comprising a completely deleted U3 region wherein said deleted U3 region is replaced by a polylinker sequence, followed by the R and U5 region. 3. A retroviral vector according to claim 1, wherein said polylinker sequence carries at least one unique restriction site. 4. A retroviral vector according to claim 3, wherein said polylinker sequence contains at least one insertion of a heterologous DNA fragment. 5. A retroviral vector according to claim 4, wherein said heterologous DNA fragment is selected from one or more elements of the group consisting of regulatory elements and promoters. 6. A retroviral vector according to claim 5, wherein said regulatory elements and promoters are target cell specific in their expression. 7. A retroviral vector according to claim 6, wherein said target cell specific regulatory elements and promoters are selected from one or more elements of the group consisting of WAP, MMTV, b-lactoglobuiin and casein specific regulatory elements and promoters, pancreas specific regulatory elements and promoters including carbonic anhydrase II and b-glucokinase regulatory elements and promoters, lymphocyte specific regulatory elements and promoters including immunoglobulin and MMTV lymphocytic specific regulatory elements and promoters and MMTV specific regulatory elements and promoters conferring responsiveness to glucocorticoid hormones or directing expression to the mammary gland. 8. A retroviral vector according to anyone of claims 5 to 7, wherein said regulatory elements and promoters regulate the expression of at least one of the coding sequences of said retroviral vector. 9. A retroviral vector according to anyone of claims 1 to 8, wherein said LTR regions are selected from at least one element of the group consisting of LTRs of MLV, MMTV, MSV, SIV, HIV, HTLV, FIV, FeLV, BLV and MPMV. 10. A retroviral vector according to anyone of claims 1 to 9, wherein said retroviral vector is a BAG vector. 11. A retroviral vector according to anyone of claims 1 to 10, wherein said coding sequence is selected from one or more elements of the group consisting of marker genes, therapeutic genes, antiviral genes, antitumour genes, cytokine genes. 12. A retroviral vector according to claim 11 , wherein said marker or therapeutic gene is selected from one or more elements of the group consisting of b- gaiactosidase gene and neomycin gene, Heφes Simplex Vims, thymidine kinase gene, puromycin gene, cytosine deaminase gene, hygromycin gene, secreted alkaline phosphatase gene, guanine phosphoribosyl transferase (gpt) gene, alcohol dehydrogenase gene and hypoxanthine phosphoribosyl transferase (HPRT) gene. 13. A retroviral vector according to anyone of claims 1 to 12, wherein at least one of said coding sequences for a retroviral protein is altered or at least partially deleted. 14. A retroviral vector according to anyone of claims 1 to 13, wherein retroviral sequences involved in integration of retroviruses are altered or at least partially deleted. 15. A retroviral vector according to anyone of claims 1 to 14, wherein said heterologous DNA fragment is homologous to one or more cellular sequences or a part thereof. 16. A retroviral vector according to anyone of claims 1 to 15, wherein said regulatory elements are regulatable by transacting molecules. 17. A retroviral vector system comprising a retroviral vector according to anyone of claims 1 to 16 as a first component; and a packaging cell line harbouring at least one retroviral or recombinant retroviral construct coding for proteins required for said retroviral vector to be packaged. 18. A retroviral vector system according to claim 17 wherein the packaging cell line harbours retroviral or recombinant retroviral constructs coding for those retroviral proteins which are not encoded in said retroviral vector according to anyone of claims 1 to 16. 19. A retroviral vector system according to claim 17 or 18 wherein the packaging cell line is selected from the group consisting of psi-2, psi-Crypt, psi-AM, GP+E-86, PA317 and GP+envAM-12. 20. A therapeutical or non-therapeutical method for introducing homologous or heterologous nucleotide sequences into human or animal cells in vitro and in vivo comprising transfecting a packaging cell line of a retroviral vector system according to anyone of claims 17 to 19 with a retroviral vector according to anyone of claims 1 to 16, and infecting a target cell population with said recombinant retroviruses produced by the packaging cell line. 21. A therapeutical or non-therapeutical method according to claim 20, wherein the nucleotide sequences are selected from one or more elements of the group consisting of genes or parts of genes encoding for proteins, regulatory sequences and promoters. 22. A retroviral provims produced by replicating the retroviral vector of anyone of claims 1 to 16 in a retroviral vector system according to anyone of claims 17 to 19 wherein said polylinker and any sequences inserted in said polylinker in the 3'LTR become duplicated during the process or reverse transcription in the infected target cell and appear in the 5'LTR as well as in the 3'LTR of the resulting provirus. 23. Use of a retroviral vector according to anyone of claims 1 to 16 for producing a pharmaceutical composition for gene therapy in mammals including humans. 24. Use of a retroviral vector system according to anyone of claims 17 to 19 for producing a pharmaceutical composition for gene therapy in mammals including humans. 25. Use of a retroviral provims according to claim 22 for producing a pharmaceutical composition for gene therapy in mammals including humans. 26. Use of a retroviral vector according to anyone of claims 1 to 16 for targeted integration in said homologous cellular sequences. 27. Use of a retroviral vector system according to anyone of claims 17 to 19 for targeted integration in said homologous cellular sequences. 28. Use of a retroviral provims according to claim 22 for targeted integration in said homologous cellular sequences. 29. mRNA of a retroviral provims according to claim 22. 30. RNA of a retroviral vector according to anyone of claims 1 to 16. 31. Recombinant retroviral particle obtained by transfecting a packaging cell line of a retroviral vector system according to anyone of claims 17-19 with a retroviral vector according to anyone of claims 1 to 16, and culturing the cells under suitable conditions. 32. Pharmaceutical composition containing a therapeutically effective amount of a recombinant retroviral particle according to claim 31.

Inventors: GUENZBURG WALTER HENRY, SALLER ROBERT MICHAEL, GUENZBURG, WALTER, HENRY, SALLER, ROBERT, MICHAEL
Applicants: GSF FORSCHUNGSZENTRUM UMWELT, GUENZBURG WALTER HENRY, SALLER ROBERT MICHAEL, GSF-FORSCHUNGSZENTRUM FUER UMWELT UND GESUNDHEIT GM, GUENZBURG, WALTER, HENRY, SALLER, ROBERT, MICHAEL
Application year: 1995
Main classification: C12N15
IPC codes: A61K35, A61K48, C12N7, C12N15