KR100527096B1 - Visa-Inactivated Targeted Expression Retrovirus Vectors - Google Patents

Abstract

The present invention provides a 5 'LTR region of U3-R-U5 structure; One or more sequences selected from coding and non-coding sequences; And a retroviral vector consisting of a 3 'LTR region consisting of R and U5 subsequent to a fully or partially deleted U3 region replaced by a polylinker sequence and performing a promoter conversion. Such retroviral vectors are useful as gene delivery vehicles for performing promoter conversion and for targeted gene therapy.

Description

Non-self-inactivated, Targeted Expression Retrovirus Vectors

The present invention relates to retroviral vectors comprising a vector (ProCon vector) on which promoter conversion is performed. The present vector system is useful as a gene delivery vehicle for targeted gene therapy.

Background of the Invention

The use of retroviral vectors for gene therapy has received a great deal of attention and has recently been one method for therapeutic gene transfer in various approved protocols in the United States and Europe (Kottani et al., 1994). Most of these protocols, however, require that infection of target cells with a retroviral vector carrying a therapeutic gene occurs in vitro and then successfully returns the infected cells to the diseased individual (Rosenberg et al., 1992; See Anderson, 1992). Such ex vivo gene therapy protocols are ideal for the modification of medical conditions in which target cell populations can be easily isolated (eg lymphocytes). In vitro infection of target cells also allows the administration of large amounts of concentrated virus, which can be rigorously tested for stability before use.

Unfortunately, only some of the possible applications for gene therapy include target cells that can be easily isolated, cultured and then reintroduced. In addition, the complex technology and associated high costs of gene therapy in vitro preclude practically widespread its use. Easier and more costly gene therapy will require in vivo access where the viral vector, or cells that produce the viral vector, are administered directly to the patient in the form of an infusion or a simple transplant of cells that produce the retroviral vector.

Of course, this kind of in vivo approach introduces a number of new problems, and above all, focus on safety considerations. The virus will probably be produced from a transplant of virus producing cells and there will be no opportunity to pre-screen the generated virus. In addition to efforts to produce new systems that minimize these risks, the recognition of the limited risks involved in the use of such systems is important.

Retroviral vector systems consist of two components (Figure 1):

1) Retroviral vectors themselves are modified retroviruses (vector plasmids) in which genes encoding viral proteins have been replaced by therapeutic genes and marker genes delivered to target cells. Viral proteins must be rescued by a second component in the system that supplies the lost virus protein to the modified virus because replacement of the encoding gene effectively lost the function of the virus.

The second component is:

2) Cell lines that produce large amounts of viral proteins but lack the ability to produce replication soluble viruses. This cell line is known as a packaging cell line and consists of a cell line transfected with a second plasmid that carries a gene that allows the modified retroviral vector to be packaged.

To generate a packaged vector, the vector plasmid is transfected into the packaged cell line. Under these conditions, the modified retroviral genome comprising the inserted therapeutic and marker genes is transcribed from the vector plasmid and packaged into modified retrovirus particles (recombinant virus particles).

This recombinant viral vector genome and all the transported label or therapeutic genes are then allowed to infect the target cells causing integration into the DNA of the target cell. Cells infected with such recombinant virus particles are unable to produce new vector viruses because there are no viral proteins present in these cells. However, the DNA of the vector carrying the therapeutic and marker genes is integrated into the DNA of the cell and can now be expressed in infected cells.

Essentially random integration of the provirus forms of the retroviral genome into the genome of infected cells (Barmus, 1988) has led to the identification of numerous cellular prototype-tumor genes by their insertional activation. (Barmus, 1988; Van Lohuichen and Bernz, 1990). The possibility that similar mechanisms cause cancer in patients treated with retroviral vectors carrying therapeutic genes intended to treat other pre-existing medical conditions has raised moral issues. Most researchers agree that replication-defective retroviral vectors such as those currently in use are very unlikely to integrate into or near cellular genes involved in cell proliferation regulation. However, the explosive expansion of individuals of replication-soluble retroviruses from a single infection will ultimately provide sufficient integration to make such phenotypic integration very likely.

Retroviral vector systems are optimized to minimize the chance of the presence of replication soluble viruses. However, recombination between the components of a retroviral vector system may lead to the development of pathogenic replication-soluble viruses. It has been well documented that populations of numerous vector systems have been constructed to minimize this risk of recombination (see Salmon and Queensburg, 1933).

Further consideration of the use of gene therapy in vivo from a safety point of view and from a purely practical point of view is the targeting of retroviral vectors. Therapeutic genes carried by the vector should be expressed indiscriminately in all tissues and cells. This is particularly important if the gene to be delivered is a toxic gene intended to eliminate specific tumor cells. Others, ie removal of non-target cells, would certainly be undesirable.

Numerous retroviral vector systems have already been disclosed that allow for the targeting of transported therapeutic genes (Salmon and Kunzburg, 1993). Most of these approaches include the use of heterologous promoters to induce the expression of heterologous therapeutic or marker genes that include the restriction of infection in predetermined cell forms or linked to specific cell forms. Heterologous promoters are used in which the promoter causes expression of linked genes only in normally active cell types. These promoters are pre-inserted in place of the gag , pol or env gene in the body of the retroviral vector in conjunction with the marker or therapeutic gene.

Retroviral long terminal repeats (LTRs) adjacent to these genes carry retroviral promoters, which are generally non-specific because they cause expression in many different cell types (Mager, 1990).

Like the tissue specific promoters mentioned above, promoter interference between the LTR promoter and heterologous internal promoters has been reported. In addition, it is known that retroviral LTRs contain strong enhancers that can affect the expression of cellular genes, either independently of or in conjunction with the retroviral promoter, near the integration site of the retroviral promoter.

It has been found that this mechanism contributes to tumorigenesis in animals (van lohui and iii). Both observations promoted the development of spontaneous-inactivation-vectors (SINs) in which retroviral promoters were functionally inactivated in target cells (PCT WO 94/29437). More modifications of these vectors include the insertion of promoter gene cassettes within the LTR region to create double replication vectors (PCT WO 89/11539). However, in these two vectors, heterologous promoters inserted into the body or LTR region of the vector are directly linked to the label / therapeutic gene.

The above-mentioned SIN vector carrying a deleted 3 'LTR (PCT WO 94/29437) is a retroviral 5' LTR promoter (U3-free 5 'LTR) for inducing expression of vector constructs in the packaging cell line. Instead, it uses a strong heterologous promoter such as that of cytomekalovirus (CMV). Heterologous polyadenylation signals are also included in the 3 'LTR (PCT WO 94/29437).

It is an object of the present invention to construct novel retroviral vectors that can be used as safe gene transfer carriers for targeted gene therapy that are less likely to recombine with packaged constructs. This novel vector replicates after infection and is transcribed into 5 'LTR in target cells, 3' LTR, inserted directly into the body of the vector rather than directly linked to the promoter, ultimately regulating the expression of the label / therapeutic gene. Carries heterologous promoters and / or regulatory factors in the

This vector does not spontaneously-deactivate and instead performs promoter exchange, giving it the name Pro Con for Promoter Conversion.

Since promoter conversion does not result in spontaneous-inactivation, the retroviral vector will be transcriptionally activated in the target cell. However, two LTRs will constitute a broad range of heterologous promoter / enhancer sequences in the target cell. This would reduce the likelihood of the integrated vector in the target cell, subject to the same inactivation over a long period of time as mentioned in the conventional vectors (Shu et al., 1989), while also increasing the safety of the system, while also preventing toxic replication soluble viruses. It will reduce the chance of recombination with the resulting endogenous retroviral sequence.

In the present invention, the 5 'LTR of the retroviral vector construct is not modified, and the expression of the viral vector in the packaged cells is caused by the normal retroviral U3 promoter. Normal retroviral polyadenylation is allowed and no heterologous polyadenylation signal is included in the 3 'LTR. This is important for the development of gene therapy strategies in vivo. Because, while packaged cells produce recombinant viruses, normal physiological control of the virus, including the normal viral control of polyadenylation and also through normal viral promoters, will prevail for a long time in vivo.

Further modification of this novel retroviral vector predicts the inclusion of cellular sequences in place of heterologous promoters and / or regulatory factors. This allows for higher selectivity for site specific recombination into cellular sequences to target the integration of retroviral vectors into specific sites in the host cell genome (Saler, 1994).

In order to achieve the above-mentioned other objects, the present invention provides a promoter conversion and 5 'LTR region of the U3-R-U5 structure; One or more sequences from coding and non-coding sequences; And a 3 'LTR region consisting of a fully or partially deleted U3 region in which the deleted U3 region is replaced by a polylinker sequence, and a 3' LTR region consisting of a contiguous R and U5 region.

The polylinker sequence carries at least one unique restriction site and preferably contains the insertion of at least one heterologous DNA fragment. The heterologous DNA fragment is preferably a DNA fragment which is selected from regulatory factors and promoters, preferably present in a target cell specific for their expression, and which also has no regulatory function.

The heterologous DNA fragment is preferably homologous to one or more cellular sequences. Regulatory factors and promoters can preferably be controlled by the processing molecule.

Still other objects, features and advantages will be apparent from the following description of the preferred embodiments of the invention.

Target cell specific regulators and promoters include Whey Acidic Protein (WAP), mouse breast cancer virus (MMTV), β-lactoglobulin and casein specific regulators and promoters, deacidification dehydratase Pancreatic specific regulators and promoters, including II and β-glogucokinase modulators and promoters, lymphocyte specific regulatory factors and promoters including immunoglobulin and MMTV lymphocyte specific regulators and promoters and glycocorticoids It is selected from one or more factors in the group consisting of MMTV specific regulators and promoters that render hormones responsive or induce mammary gland expression. The regulators and promoters preferably regulate the expression of at least one of the coding sequences of the retroviral vector. LTR regions include murine leukemia virus (MLV), mouse breast cancer virus (MMTV), murine tumor virus (MSV), monkey immunodeficiency virus (SIV), human immunodeficiency virus (HIV), feline leukemia virus (FELV), bovine leukemia virus (BLV) and LTRs of perennial-piper-monkey virus (MPMV).

Retroviral vectors are preferably based on BAG vectors (Price et al., 1987) or LXSN vectors (Miller and Dozman, 1989).

The coding sequence is preferably selected from one or more factors of the group consisting of marker genes, therapeutic genes, antiviral genes, antitumor genes, cytokine genes.

The label and therapeutic genes are preferably β-galactosidase gene, neomycin gene, herpes simple virus thymidine kinase gene, puromycin gene, cytosine deaminase gene, hinolomycin gene, secreted alkaline phosphatase Gene, guanine phosphoribosyl transferase (gpt) gene, alcohol dehydrogenase gene, and hyposantiphosphoribosyl transferase (HPRT) gene.

Another embodiment of the invention illustrates the replacement or partial deletion of at least one retroviral sequence required for integration of retroviruses.

In an embodiment of the invention the retroviral vector system, as mentioned above, comprises a retroviral vector as a first component and at least one retrovirus or recombinant retrovirus encoding a protein required for the retroviral vector to be packaged. It is constructed and provided as a packaged cell line for mounting the construct.

The packaging cell line is equipped with a retrovirus or recombinant retroviral construct that encodes unencoded retroviral proteins in the retroviral vector. The packaging cell line preferably allows expression of surface proteins from psi-2, psi-Crypt, psi-AM, GP + E-86, PA317 and GP + env AM-12, or other enveloped viruses. Is selected from all factors super-infected with recombinant constructs.

Another embodiment of the present invention involves the use of a packaging cell system that subsequently harbors a recombinant retroviral construct that lacks integrase function. As mentioned above, the retroviral vector of the present invention is introduced into a retroviral packaging cell line and, after infection of the target cell, is provided to the provirus of the retrovirus, as mentioned above, wherein the polylinker at 3 ′ LTR. And the sequence inserted into the polylinker is replicated during the process of reverse transcription in infected target cells and appears in the 5 'LTR as well as in the 3' LTR of the resulting provirus.

The invention also includes proviral mRNA of the retrovirus according to the invention and RNA derived from the retroviral vector according to the invention.

In addition, embodiments of the present invention comprises the steps of transfecting the packaging cell system of the retroviral vector system according to the present invention with the retroviral vector according to the present invention and infecting the target cell subjects with a recombinant retrovirus produced by the packaging cell system. Non-therapeutic methods for introducing homologous and / or heterologous nucleotide sequences into human or animal cells, which are constructed, in vitro and in vivo, are provided. The nucleotide sequence is selected from one or more factors in the group consisting of genes encoding protein, regulatory sequences and promoters and portions of genes.

Retroviral vectors, retroviral vector systems and retrovirus proviruses as well as RNA are used to produce pharmaceutical compositions for gene therapy in mammals, including humans. In addition, they are used for targeted integration into homologous cellular sequences.

Promoter Switch

The present invention utilizes the principle of promoter conversion typical for retroviruses. The retroviral genome consists of RNA molecules with the structure R-U5-gag-pol-env-U3-R (Figure 2). During the reverse transcription process, the U5 region is located at the right hand end of the cloned and produced DNA molecule, while the U3 region is located at the left hand end of the cloned and produced DNA molecule (FIG. 2). The resulting U3-R-U5 structure is called LTR (long terminal repeat) and is therefore either repeated at both ends of the DNA structure or consistent with the provirus (Barmus, 1988). The U3 region is equipped with a promoter at the left hand end of the pro virus (see below). This promoter induces the synthesis of RNA transcripts starting at the border between the left hand U3 and R regions and ending between the right hand R and U5 regions (FIG. 2). This RNA is packaged into retroviral particles and transported to the target cell to be infected. The RNA genome in the target cell is reverse transcribed again as mentioned above.

Retroviral vectors are constructed according to the invention such that the right-hand U3 region is altered, but the normal left-hand U3 structure is maintained (FIG. 3); This vector can be normally RNA transcribed using a normal retroviral promoter located within the left-hand U3 structure (FIG. 3). However, the resulting RNA will only contain altered right-hand U3 structures. In infected target cells, after reverse transcription, this altered U3 structure will be located at both ends of the retroviral structure (FIG. 3).

If the altered region carries a polylinker (see below) instead of the U3 region, all promoters can be easily inserted, including those that induce tissue specific expression, such as the WAP promoter (see below). This promoter will then be widely used in target cells for the expression of the linkage gene carried by the retroviral vector. Alternatively or additionally, DNA fragments homologous to one or more cellular sequences can be inserted into the polylinker for the purpose of gene targeting, by homologous recombination (see below).

According to the present invention the term "polylinker" refers to a short stretch of artificially synthesized DNA with a number of unique restriction sites that allow for easy insertion of a promoter or DNA fragment. The term “heterologous” refers to any combination of DNA sequences that is not normally found in nature.

Gene expression is regulated by promoters. Without the function of the promoter the gene would not be expressed. Normal MLV retroviral promoters are not selective because they are active in most cell types (Mager, 1990). However, there are numerous promoters that are active only in very specific cell types. Such tissue-specific promoters would be ideal for the regulation of gene expression in retroviral vectors, ie, for the limited expression of therapeutic genes in specific target cells.

Expression of retroviral vectors in the packaging cell line is regulated by normal non-selective retroviral promoters contained in the U3 region (FIG. 3). However, as soon as the vector enters the target cell, promoter conversion occurs and, for example, a therapeutic or marker gene such as b-galactosase is expressed from a tissue specific promoter introduced into the polylinker (FIG. 3). Not only can virtually all tissue specific promoters be included in the system, but if selective targeting of many different cell types is provided, and additionally following conversion, the structure and properties of the retroviral vector are no longer relevant to the virus. It will not be similar to that. This is of course a very important result from the safety point of view. Because the situation of artificial retroviral vectors is actually willing to genetically recombine with retroviral packaging constructs and / or endogenous retroviruses to produce toxic viruses. Pro Con vectors are not similar to retroviruses. Because they no longer carry the U3 retrovirus promoter, which reduces the likelihood of genetic recombination after conversion.

Retroviral promoter structures are carried within the U3 region of the LTR. LTRs carry signals that allow them to integrate into the genome of the target cell. Integration of retroviral proviruses may also contribute to toxic transformations (Van Rohuichen and Wong, 1990). In one embodiment of the present invention, the Pro Con vector may carry modified LTRs that no longer carry the signals required for integration. Again this increases the potential stability of these vector systems.

Gene targeting

According to another aspect of the invention, retroviral vectors are used for targeted integration into target cells. The integration of the proviral DNA version of the retroviral genome into the target cell is a major advantage in the use of retroviruses as vectors when compared to other viruses such as adenoviruses. This is because it allows long-term stable expression of the delivered gene. However, the random nature of this integration also represents a major disadvantage to the use of retroviral vectors. This is because it increases the likelihood of interstitial (non) activation of cellular tumor suppressor genes or proto-tumor genes and the induction of tumors (Van Rohuichen and Bernz, 1990).

Homologous recombination has been successfully used for the integration of transfected or microinjected DNA into specific DNA sites and is commonly used to reduce "knock-out" transgenic mice or animals (see café). , 1989; Bradley et al., 1992; Morrow and Cukerrapati, 1993). Unfortunately, the efficiency of DNA delivery by such simple physical methods is very low. In contrast, retrovirus mediated gene transfer is highly efficient, ie infectious in individuals of nearly 100% of cells. Combination of retroviral gene delivery with homologous recombination will allow construction of a system that is ideal for site-targeted integration.

We have investigated the possibility of introducing long homologous DNA fragments into retroviral vectors at different locations to enhance integration by homologous recombination (Seller, 1994). Both gene conversion and homologous recombination were evaluated. Using a cell line carrying a single copy of the HSV-tk gene as a target, we were able to disintegrate the target 15 times higher than previously reported by others (Ellis and Bernstein, 1989). Cloning of the recombinant fragment of DNA confirmed the presence of both the target tk sequence and the retroviral vector (Seller, 1994).

DNA fragments homologous to the cellular sequence for targeted integration are inserted into the polylinker of the Pro Con vector. After infection and reverse transcription of the target cell, these sequences will appear at the 5 'end of the provirus. Terminal homology has been shown to promote homologous recombination (Bradley, 1991) to homologous cellular sequences. Infection of target cells carrying a mutated form of homologous sequence will result in recombination and repair of the mutated sequence. Only homologous sequences will be bound to the cellular genome or a complete vector will be inserted (Seller, 1994). Not only does this vector type have the potential for use in gene repair, it can also be used to cause the integration of retroviral vectors carrying therapeutic genes at specific locations in the genome that do not carry active genes. This will significantly reduce the likelihood of invasive activation or inactivation as mentioned above and will contribute to the safety of the use of retroviral vectors.

The following examples will further illustrate the invention. However, these embodiments are not intended to limit the scope of the invention as the formulas will be clear, and other formulas and substitutions will be apparent to those skilled in the art.

Recombinant DNA methods used in the present invention are standard procedures well known to those skilled in the art and are described in detail in, for example, "Molecular Cloning" (Samburg et al., 1989) and "Performance Guide to Molecular Cloning" (Perval, 1984). It is.

Brief description of the drawings mentioned in the following examples:

1; Retrovirus Vector System

2; Retrovirus genome, reverse transcription

3; Pro Con principle

4; PCR analysis, MLV probe

5; PCR analysis, MMTV probe

6; Β-galactosidase expression in infected NIH and EF43 cells

7; Β-galactosidase Expression in First Mammary Cells Infected from Pregnant Mice

8; Β-galactosidase Expression after Mammary Injection into Mammary Gland and Skin of Pregnant Balb / c Mice

9; Β-galactosidase Expression in Infected Breast Cancer Cells

10; Targeted integration of retroviral vectors by homologous recombination

Example 1

The β-galactosidase gene in the murine leukemia virus (MLV) retroviral vector, known as mammary gland specific expression BAG after infection with a Pro Con vector carrying a breast-specific promoter, is irregular in the U3 region of the LTR. Induced by (ie, non-tissue specific) MLV promoter (FIG. 3).

According to the present invention a defective BAG vector constructed with a missing MLV promoter (U3) located in the 3 'LTR and replaced with a polylinker that allows for easy introduction of heterologous promoters. BAG vectors lacking U3 are expressed from the MLV promoter (U3) in the 5 'LTR when introduced into the packaging cell line. As a result of the rearrangement that occurs in the retroviral genome during its life cycle following infection of its target cell, the polylinker will replicate at both ends of the retroviral genome as mentioned above. Thus a retroviral vector can be constructed in which the expression of the β-galactosidase gene of BAG is regulated by all heterologous promoters inserted into the polylinker in the target cell (FIG. 3).

In accordance with the principles mentioned above, the following specific promoters were inserted into the polylinker region or the modified BAG vector.

Some subregions of the mouse breast cancer virus (MMTV) promoter include regions that are responsive to glucocorticoid hormones and regions that contain factors that allow expression in the mammary gland.

Whey protein (WAP) promoters regulate the expression of WAP and are produced only in the mammary glands of pregnant and lactating rodents.

Modulation of β-galactosidase gene expression by promoters inserted into polylinkers was confirmed by infection studies using constructed MMTV and WAP retroviral vectors to infect various cells.

To generate retroviral vector particles, MMTV and WAP Pro Con vectors were transfected with the packaging cell line GP + E86 (Marcowitz et al., 1988). After the selection of neomycin resistance encoded by the vector, stable individuals and clones of the cells producing the recombinant Pro Con virus were obtained.

Viral supernatant from these individuals was used to infect the mouse mammary cell line EF43 (Queensberg et al., 1988) as well as the mouse fibroblast cell line (Zeinchl et al., 1969). Four days after infection, the target cells were lysed and the quantitative β-galactosidase assay showed no expression in both cell types infected by the Pro Con vector carrying WAP and in both cell types from the Pro Con vector carrying MMTV. Good expression was revealed (FIG. 6). The result is that the WAP promoter only acts in vivo during late gestation and lactation and does not function in a simple in vitro breast cell culture system as shown by EF43 cells. To investigate whether the WAP carrying Pro Con vector is activated in a cell culture system derived from a complex initial breast, firstly tissues from mice (Fig. 7) or breast cancer (Fig. 9) with a gestation period of 8-10 days (Fig. organoids) were cultured and infected with supernatants from the same stably transfected population of the transfected cell line. Both Pro Con vectors carrying WAP and MMTV promoter fragments were active in these first cells (FIG. 7) and in breast cancer-derived cells (FIG. 7) as indicated by β-galactosidase activity.

To investigate whether WAP and MMTV carrying Pro Con vectors were active in vivo and expression of β-galactosidase was restricted to mammary gland in vivo, the medium containing the recombinant Pro Con virus was in situ. situ) is injected into the mammary gland and skin of mice that are 8-10 days pregnant. After 5 days the mice were sacrificed, cell extracts were prepared and β-galactosidase assay was performed. Both Pro Con vectors carrying WAP and MMTV cross sections were expressed only in the pregnant mammary gland and not in the epidermis (M and S in FIG. 8). So in vivo regulatory factors from both promoters limit expression into the mammary gland, whereas in vitro regulatory factors from the WAP promoter retain their stringent tissue specificity, but not those of MMTV.

These Pro Con vectors carrying tissue specific promoters and regulatory factors are useful for inducing expression of therapeutic genes in predetermined cell morphology, regulation and organs. Potential therapeutic genes include melittin with anti-HIV and anti-tumor effects, and thymidine kinase, guanine phosphory, as well as genes involved in regulating cellular circulation such as SCI / WAF-1 / CIP-2. Genes that are lethal to cells, including bocitic transferase and cytosine deaminase genes, cytochrome P450.

Example 2

Confirmation of promoter conversion in cells infected with the Pro Con vector that initially carried the MMTV promoter at 3 'LTR.

Pro Con vectors carrying a promoter from mouse breast cancer virus (MMTV) were transfected into the packaging cell line and the resulting recombinant vector particles infected the established human bladder cancer cell line (EJ). Infected cell clones were selected in media with neomycin analog G418 (because the vector carries a neomycin resistance gene derived from the internal SV4O promoter). DNA was produced from one of the infected clones and untransfected parental EJ cells, which DNA was used for polymerase chain reaction (PCR). PCRs are specifically recognized and either of the two primers that bind to the MMTV sequence (A, B in FIGS. 4 and 5) or the MLV R region (C in FIG. 4), with the primers located within the label gene (FIGS. 4 and 5). ) Was performed. Since marker gene primers are only located downstream of MMTV (MLV R region), if promoter conversion occurs, a positive PCR signal obtained with MMTV primers in combination with marker gene primers suggests this. In Figure 4 PCR products using primers A, B or C are shown after hybridization with labeled fragments from the MLV sequences, confirming that all three PCR products are MLV derived. The size of the fragments demonstrates that promoter conversion has taken place. 5 shows PCR products using primers A or B and hybridized with MMTV specific probes, again confirming that promoter conversion occurred.

Example 3

Construction of Pro Con Vectors for Targeted Integration

If a BAG vector as mentioned in Example 1 is used, a retroviral vector capable of inserting a DNA sequence homologous to the cellular sequence into the LTR can be constructed. The resulting vector can be used to target the integration of all or a portion of the inserted vector into a homologous sequence inserted into the vector or a homologous sequence present in the host cell genome.

According to the principles mentioned above, fragments of the thymidine kinase (+ k) gene of herpes simple virus (HSV) were inserted into the polylinker region of the modified BAG vector (tk variant, seller, 1994 in Figure 10).

A cell line has also been established that does not have a functional copy of the mammalian tk gene but instead carries one copy of the HSV-tk gene (Seller, 1994). This cell line was infected with BAG vectors carrying tk and cells were selected that disrupted the HSV-tk gene (FIG. 10).

The above examples illustrate the principles and results of promoter conversion vectors provided by the present invention.

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Claims (23)

(a) 5 'LTR region of U3-R-U5 structure; (b) one or more sequences selected from coding and non-coding sequences to be inserted into the vector body; And (c) a retroviral vector carrying a promoter conversion comprising a 3 ′ LTR region comprising a U3 region that is completely or partially deleted and does not contain the original retrovirus derived promoter sequence, In nature, a retrolinker comprising a polylinker sequence containing a heterologous promoter not found in the retrovirus is inserted into the deleted U3 region, wherein the promoter regulates expression of the one or more coding sequences after target cell infection. Virus vector. The method of claim 1, And said polylinker sequence has at least one specific restriction enzyme recognition site. The method according to claim 1 or 2, Retroviral vector, characterized in that the polylinker sequence further comprises a target cell specific regulator. The method according to claim 1 or 2, Wherein said heterologous promoter and regulator have target cell specificity upon their expression. The method of claim 4, wherein The promoters and modulators are pancreatic specific regulators and promoters, including WAP, MMTV, β-lactoglobulin and casein specific regulators and promoters, deacidase II and β-glucokinase modulators and promoters, immunoglobulins and MMTVs. A retroviral vector, characterized in that it is selected from the group consisting of lymphocyte specific regulators and promoters, including lymphocyte specific regulators and promoters, and MMTV specific regulators and promoters. The method according to claim 1 or 2, The LTR region is at least one factor selected from the group consisting of LTR regions of MLV, MMTV, MSV, HIV, SIV, HIV, HTLV, FIV, FeLV, BLV and MPMV. The method according to claim 1 or 2, Retroviral vector, characterized in that the retroviral vector is a BAG vector. The method according to claim 1 or 2, Retroviral vector, characterized in that the coding sequence is at least one factor selected from the group consisting of a label gene, therapeutic gene, antiviral gene, anti-tumor gene, cytokine gene. The method of claim 8, The marker or therapeutic genes include β-galactosidase gene and neomycin gene, herpes simplex virus, thymidine kinase gene, puromycin gene, cytokine deaminase gene, hygromycin gene, secreted alkaline phosphatase gene, guanine A retroviral vector, characterized in that it is at least one element selected from the group consisting of phosphoribosyl transferase (gpt) gene, alcohol dehydrogenase gene, and hypoxanthine phosphoribosyl transferase (HPRT) gene. The method according to claim 1 or 2, Inclusion of said intracellular sequence instead of heterologous promoters and / or modulators in order to target insertion of retroviral vectors at specific locations within the host cell genome to increase selectivity for position specific recombination with intracellular sequences. Retrovirus vector characterized by. A retroviral vector according to any one of claims 1 to 9 and 10 as a first component; And a packaging cell line comprising at least one retrovirus or recombinant retrovirus construct encoding at least one protein selected from the group consisting of gag, pol and env required for packaging the retroviral vector. Vector system. The method of claim 11, Said packaging cell line comprises a retrovirus or a recombinant retrovirus construct which encodes a retrovirus protein which is not encoded by the retroviral vector according to any one of claims 1 to 9 and 10. Virus vector system. The method of claim 11 or 12, Retroviral vector system, characterized in that the packaging cell line is selected from the group consisting of psi-2, psi-Crypt, psi-AM, GP + 3-86, PA317 + env AM-12. For animals other than humans, the packaging cell line of the retroviral vector system according to any one of claims 11 to 13 is transformed with the retroviral vector according to any one of claims 1 to 9 and 10. Making a step; And infecting a target cell population with said recombinant retrovirus produced by the packaging cell line. 18. A therapeutic method for introducing homologous or heterologous nucleotide sequences into an animal cell in vivo . The method of claim 14, Wherein said nucleotide sequence is one or more factors selected from the group consisting of genes or portions thereof, regulatory sequences and promoters encoding proteins. A retroviral provirus generated by replicating a retroviral vector according to any one of claims 1 to 9 and 10 in a retroviral vector system according to any one of claims 11 to 13, The polylinker in the 3 'LTR and the sequence inserted into the polylinker are replicated during processing or reverse transcription in infected target cells and are present in the 5'LTR as well as the 3'LTR of the resulting provirus Proviruses of retroviruses. A packaging cell line of the retroviral vector system according to any one of claims 11 to 13 is transformed into a retroviral vector according to any one of claims 1 to 9 and 10 and the cells are subjected to appropriate conditions. Recombinant retrovirus particles, characterized in that obtained by culturing. The retroviral vector according to any one of claims 1 to 9 and 10, the retroviral vector system according to any one of claims 11 to 13, the retroviral provirus according to claim 16 and / or Or a pharmaceutical composition comprising a therapeutically effective amount of a recombinant retroviral particle according to claim 17. The method of claim 21, A pharmaceutical composition, characterized in that it is used for antiviral and / or anticancer gene therapy. The method of claim 18, A pharmaceutical composition, characterized in that it is used to target insertion into said homologous intracellular sequence for gene repair or to transport said therapeutic gene to a specific location in the genome known to contain a therapeutic gene. MRNA of the retroviral provirus according to claim 16. RNA of the retroviral vector according to any one of claims 1 to 9 and 10. Transforming the packaging cell line of the retroviral vector system according to any one of claims 11 to 13 with the retroviral vector according to any one of claims 1 to 9 and 10; And infecting a target cell population with said recombinant retrovirus produced by the packaging cell line. A non-therapeutic method for introducing homologous or heterologous nucleotide sequences into an animal cell in vitro .

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