WO1988008454A1 - Process for amplifying expression and transmission of cloned genes in eukaryotic cells - Google Patents

Abstract

A process for amplifying expression and transmission of cloned genes by introducing a replication defective retroviral expression vector into a mixture of cells that includes two eukaryotic packaging cell lines that package virions with different host ranges. Virions produced by the cells of one of the cell lines can infect the cells of the other cell line. Cross infection continues in an exponential ''ping-pong'' fashion that allows superinfection of both cell lines. The packaging cells express the cloned gene in relatively large and commercially useful amounts, and produce high titers of virions. The high titer of virions produced by this process can also be used to infect target cells for producing commercially useful quantities of gene product.

Description

PROCESS FOR AMPLIFYING EXPRESSION AND TRANSMISSION OF CLONED GENES IN EUKARYOTIC CELLS This invention was made with government support under Grant No. 5R01 CA 25810, awarded by the National Institutes of Health. The government has certain rights in this invention.

The present invention concerns a process for amplifying the expression and transmission of cloned genes in eukaryotic cell cultures using retroviral vectors.

It is often advantageous to express gene products in higher eukaryotic or mammalian cells to avoid processing defects that characteristically occur in proteins produced by bacteria or lower eukaryotes such as yeast or insects. Higher eukaryotic or mammalian cells can be genetically altered to express such defect-free products by introducing genes of interest into the cells. One method for transferring genes into mammalian cells is DNA transfection, in which DNA is introduced into cells in culture using dextran sulfate or as part of a coprecipitate with calcium phosphate.

Unfortunately, DNA transfection is a very inefficient means of transferring genes into mammalian cells, because fewer than one in a thousand cells will stably incorporate the newly transferred gene. Identification and isolation of stable transfectant cells has therefore required complex screening procedures and use of dominant selectable marker genes. Yet another problem with DNA transfection is that not all cultured cell lines are susceptible to this method of gene transfer. In addition, different cell clones that become stably transfected with a gene express that gene to different extents. Moreover, the extent of expression is usually low. Therefore, laborious and costly screening of transfectant cell clones is generally required to identify a clone with a satisfactory level of expression. Attainable quantities of gene expression by this method have usually been insufficient for commercial use.

To overcome some of these problems, several alternate methods have recently been developed for gene transfer with cells of higher eukaryotes. One such technology is retroviral mediated gene transfer, which uses retroviruses to deliver genes into cells. Retroviruses contain an RNA genome. After infection into cells, the genomic RNA is - 2 -

used as a template to form DNA by a process called reverse transcription. This DNA is then integrated into a chromosome of the cell where it is called a provirus. Expression of this chromoso ally integrated provirus results in the synthesis of new copies of the retroviral RNA genome and in synthesis of the proteins that are encoded by the virus. If these virus-encoded proteins include the structural proteins necessary to constitute virions (these are called the viral gag, pol and env proteins), then the cell will form progeny virions that are released by budding from the cell surface. However, if the virus that infected the cells does not encode the latter viral structural proteins, then progeny virions cannot be formed. Retroviruses that lack functional gag, pol and/or env genes are called repl cation-defective because they cannot independently cause the synthesis of their own progeny virions. Replication-defective retroviruses are best understood by contrasting them with replication-competent viruses. The replication competent retroviruses (also called "helper" viruses) contain two types of information that are classified as trans functions and cis functions. The trans functions are the gag, pol, and env genes that encode the structural proteins necessary to constitute progeny virions. The cis functions are sequences in the retrovirus that enable the genomic RNA to become incorporated into released virions and to become converted into functional proviral DNA by reverse transcription. Replication-defective retroviruses contain cis functions but not trans functions, and can be released from cells which contain a helper virus that encodes the gag, pol, and env virion structural proteins.

Retrovirus preparations that contain a cloned gene of interest are typically obtained by a multi-step procedure that uses retroviral packaging cell lines and retroviral expression vectors. Retroviral packaging cell lines are typically formed by transfecting cells with DNA that encodes gag, pol, and env genes (i.e., the trans functions) but that lacks some or all of the requisite cis-sequences. Such packaging cells therefore contain gag, pol and env proteins but do not release infectious progeny virions. Retroviral expression vectors are typically DNA plasπrids that lack functional gag, pol and 54

- 3 -

env genes but that contain the c _s sequences that are characteristic of transmissible retroviral nucleic acids. In addition, these vectors contain one or more sites for insertion of cloned genes of interest. The gene of interest (i.e., any nucleic acid that can be inserted into a retroviral vector, including a cDNA or a modified or synthetic gene rather than a normal gene with introns) is commonly introduced into the retroviral expression vector by standard cloning techniques. The resulting vector that contains the cloned gene is typically introduced into a retroviral packaging cell line by DNA-mediated transfection. The rare retroviral packaging cells that stably incorporate this vector then begin to synthesize the genomic RNA encoded by the vector, to express the cloned gene of interest that was inserted into the vector, and to release "helper-free" virions that contain the genomic vector RNA. Because the latter genomic RNA contains proper c s sequences, the latter virions are infectious and they can be used to infect cells of interest to an investigator, hereinafter called target cells. The infected target cells acquire a copy of the provirus in their chromosomes and stably express the cloned gene. However, these infected target cells do not release progeny virions because the retroviral vector lacks trans functions. Although retroviral gene transfer is an established method for obtaining cellular expression of a foreign gene, it has not proved to be very efficient or convenient. Packaging cells into which one has introduced a retroviral expression vector with an inserted cloned g_ene usuall express the cloned gene only weakly and produce a low titer of virus (typically about 10^-10^ virions/ l). Extensive and costly screening of cell clones is generally required to identify higher expressors. Such a low titer of virions is insufficient to easily and efficiently infect target cells at a high multiplicity of virus per cell. Consequently, the target cells also typically fail to produce the desired protein in commercially desirable amounts. For these reasons, the retroviral gene transfer method is generally considered to be inappropriate for production of conτπercially useful quantities of proteins that are encoded by cloned genes. Cells that release retroviruses (e.g., retroviral packaging cell lines) must synthesize the gag, pol and env structural components that constitute the virions. The env component is a glycoprotein that occurs in the surface membranes of the cells in which it is synthesized. When a virion is produced by budding from a cell, the virion acquires its envelope membrane from the surface membrane of the cell. Simultaneously, the virus surface envelope acquires the env glycoprotein components which had been situated in the surface membrane of the cell. These env glycoprotein constituents enable the progeny virions to bind specifically to and infect cells that have functional cell surface receptors for that same env glycoprotein. However, any cell that synthesizes a specific env glycoprotein loses its receptors for that glycoprotein by a process called "interference". Because any retroviral packaging cell line must of necessity synthesize env glycoproteins (otherwise the cells would be incapable of packaging complete virions), the cells of a packaging line lack functional receptors for the virus they release.

Therefore, when a retroviral expression vector with a cloned gene is introduced into a retroviral packaging cell line by the methods of prior technology, the genomic RNA of the expression vector is synthesized to a limited extent and progeny virions are released -into the culture medium in a correspondingly limited quantity.

Moreover, the protein encoded by the cloned gene is also synthesized in a correspondingly limited quantity. Because these progeny virions with the cloned gene cannot infect any cells in the cultures in which they are formed, the virus cannot grow and replicate by infection within the culture. The virus that contains the cloned gene of interest can infect certain types of target cells in other cultures, but by the methods of prior technology this virus cannot infect any cells in the culture of packaging cells in which it is produced. Amplification of the virus by replication and infection would be desirable because the cells would thereby acquire more copies of the provirus in their chromosomes and this would result in increased expression of the cloned gene and in increased release of progeny virions by the cell. Because of interference, such amplification was completely prevented in the cultures employed by the prior technology. The present invention overcomes the foregoing problems by producing a high titer virus preparation and commercially desirable quantities of gene product. A retroviral expression vector that contains a cloned gene is introduced into a mixture of cells from two eukaryotic packaging cell lines that package virions with different host ranges. Virions produced by a first cell line can infect any cells of the second cell line, and virions produced by the second cell line can infect any cells of the first cell line. Cross-infection continues in an exponential "ping-pong" fashion between the cell types in the mixed culture until substantially all cells of the culture contain many copies of the provirus that contains the cloned gene. These cells express the cloned gene in relatively large amounts and they release correspondingly large amounts of the virions of interest. Even a small quantity of retroviral vector added to the mixed culture system suffices to initiate this process of ping-pong amplification. Because all cells in the mixed cultures spontaneously become high expressors of the gene of interest, elaborate, time-consuming and costly screening of cell clones is unnecessary.

The combination of packaging cell lines in a coculture produces a high titer virion preparation having approximately 10 virions or more per ml. The virus can then be used to infect target cells for expression of the gene of interest. The high titer virion preparation is simple to obtain and can be used to infect more target cells at a greater multiplicity of virions/cell than the low titer preparations produced by prior art technology. These are important advantages because the virions that contain the cloned gene are replication-defective and cannot independently reproduce. The greater degree and multiplicity of target cell infection that results from use of the high titer virion preparation brings about greater expression of the gene of interest.

It is therefore an object of this invention to provide a retroviral gene transfer method which amplifies expression of a gene of interest in target cells and packaging cells.

Another object is to provide such a method which increases tne titer of virus produced by packaging cells for more efficient infection of target cells. Yet another object is to provide a retroviral gene transfer method which can be used without the many complex, labor-intensive and time-consuming colony screening protocols required of prior methods. Still another object is to provide a method which does not require isolation of stable transfectants.

Even yet another object is to provide such a method in which cells with amplified expression of a cloned gene of interest remain viable and stably express the cloned gene.

Another object is to provide such a method which produces protein products that are properly processed and therefore free of defects. These and other objects and features of the invention will be apparent from the following description and accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram illustrating the retroviral life cycle in which infected cells become resistant to superinfection by the virus they release.

FIG. 2 is a schematic diagram illustrating different retroviral receptors on different types of mouse cells.

FIG. 3 is a schematic diagram illustrating the inability of a retroviral packaging cell line to become reinfected by the virions that they release.

FIG. 4 is a schematic diagram which illustrates the present invention in which mixed culture ampl fication of virions and gene products is achieved.

FIG. 5 is a photograph of immunoblots of cell lysates from mixtures of psi and PA12 cells.

FIG. 6 is an immunoblot comparing gp55 gene expression using a mixed culture method and standard methods of SFFV expression.

FIG. 7 is an immunoblot showing the transfer of high level expression of gp55 using SFFV produced in the mixed culture system of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The process of the present invention employs retroviral vectors and mixtures of retroviral packaging cell lines to amplify expression of cloned genes in cultures of vertebrate cells. By using mixtures of packaging cell lines that release retroviruses with different host ranges, the interference barriers to retroviral superinfection can be overcome and the cells of each cell line become infected with multiple copies of the virus that contains the cloned gene. The resulting packaging cells that contain many copies of the provirus synthesize the encoded protein in relatively large amounts. Because the virus potentially will become amplified indefinitely in the mixed culture system, the expression of the cloned gene will increase until its encoded protein constitutes a substantial proportion of the total protein being synthesized in the culture. A high titer of virions will also be produced by the packaging cell lines, and these virions can be used to infect target cells which in turn produce the encoded proteins. The high titer of virions causes the target cells to produce a correspondingly large quantity of the gene product of interest. Forming Retroviral Packaging Cell Lines Retroviral packaging cell lines can be formed by stably transfecting cells with DNAs that encode functional gag, pol and env proteins but that lack some or all of the requisite cis-sequences, as described in Miller et al., Molecular and Cellular Biology, 5:431-437 (1985); Mann et al, Cell, 33:153-159 (1983); and Sorge et al . , Molecular and Cellular Biology, 4:1730-1737 (1984). Replication-Defective Retroviruses

Replication-defective retroviruses generally lack functional gag, pol and/or env genes. However, they can be transmitted between cells in the presence of a repl cation-competent helper virus as described in Weiss et al., RNA Tumor Viruses, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Because replication defective retroviruses are made transmissible by the helper virus, the replication defective retroviral genomes must contain all of the cis-sequences described above that are necessary for causing their incorporation into virions and for their reverse transcription to form functional proviral DNAs.

When replication-defective retroviral genomes are introduced (e.g., by transfection or by infection) into retroviral packaging cell lines, the resulting cells can produce virion particles that contain the repl cation-defective retroviral genomic RNA. These "helper-free" virion preparations can be used to infect target cell cultures [as in Miller et al. (1985), Mann et al. (1983), and Sorge et al. (1984) above] or animals [as in Williams et al., Nature 310:476-480 (1984); Lemischka et al., Cell 45:917-927 (1986); Bestwick, et al., J. Virol 56:660-664 (1985); and Wolff and Ruscetti, Science 228:1549-1552 (1985)]. The target cells become infected and express the encoded gene products, but they do not release any progeny virus.

Retroviral expression vectors have been described by Cepko et al., Cell 37:1053-1062 (1984); Rosen et al., J. Virol. 57:379-384 (1986) and others. Basically, such vectors are plasmids that encode replication-defective retroviruses. Cloned genes of interest can be inserted into expression sites in the plasmids as in Cepko et al., 1984. After the resulting expression vectors are introduced (e.g., by DNA-mediated transfection) into retroviral packaging cell lines, the resulting cells release "helper-free" virion particles that contain the cloned gene of interest. These helper-free virions can infect cells but they do not encode the gag, pol and env trans components needed to form progeny virions. Retroviral Host-Range and Interference of Infected Cells to Superinfection Retroviruses have been classified into discrete host-range types (Weiss et al.). Although there are other factors that can contribute to the host-range properties of any retrovirus, a major contributor to the host-range is the viral env gene. Essentially the env gene encodes the virion membrane envelope glycoprotein that confers on the virus the ability to bind to receptors that occur specifically on the surfaces of susceptible cells. Cells that lack these receptors cannot be infected by the retrovirus. Therefore, the host-range of the virus is limited by its env gene and by the host-range distribution of the corresponding cell surface receptor. For example, murine leukemia viruses (MuLVs) are classified as ecotropic (able to infect only mice or rats), amphotropic (able to infect all mammalian cells), or xenotropic (able to infect all mammalian cells except mice). Each of these MuLV host-range classes uses a distinct host cell receptor for infection. The interaction between the retroviral-encoded env gene product and its receptor also causes interference to superinfection (Weiss et al.). Thus, cells that express a retroviral env gene are resistant to superinfection by any retrovirus that uses the same cell surface receptor for infection. For example, cells that synthesize an ecotropic ej glycoprotein cannot be superinfected by any ecotropic MuLV. Therefore, the retroviral host-range classes are also interference classes, and each retrovirus is a member of a particular host-range/interference family. This interference to superinfection is believed to occur because the synthesis of the retroviral env glycoprotein by a cell somehow blocks the corresponding receptor sites on that cell (Weiss et al.).

FIG. 1 schematically illustrates the concepts of retroviral host range and interference of infected cells. An uninfected cell 10 having a nucleus 12 is shown at the top of the drawing. Its surface membrane 14 contains receptors for different host-range classes of virus (drawn as solid symbols). In the drawing, the receptors 16 for Ecotropic (Eco) virus 18 and receptors 20 for Amphotropic (Ampho) virus 22 are shown. The viruses have Eco and Ampho envelope glycoproteins 24, 26 respectively. Infection 28, 30 by a replication competent virus of either host-range class (Eco or Ampho) results in synthesis by an Eco infected cell 32 or an Ampho infected cell 34 of progeny Eco or Ampho virus 36, 38, respectively. However, the infected cell also now contains on its surface the envelope glycoprotein encoded by its virus, and for this reason the infected cell lacks the corresponding receptors. Specifically, each Eco-infected cell 32 contains Eco envelope glycoproteins 24 on its surface and therefore lacks functional Eco receptors. And, each Ampho infected cell 34 lacks functional Ampho receptors because it contains Ampho envelope glycoprotein 26. The mechanism for this removal of the corresponding receptors is called "interference" but is not currently understood. Thus, a cell 32 that synthesizes Eco envelope glycoproteins 24 lacks functional Eco receptors 16 and therefore cannot be superinfected 40 by an Eco virus 18, 36. Similarly, a cell 34 that synthesizes Ampho envelope glycoproteins 26 lacks functional Ampho receptors 20 and cannot be superinfected 42 by an Ampho virus 22, 38. Although this diagram shows infection by retroviruses that _z - 9a - are replication competent (i.e., the infected cells release progeny virions 36, 38 that are also infectious), the critical factor that causes interference to superinfection is the viral envelope glycoprotein 24, 26. Thus, a cell that synthesizes retroviral envelope glycoproteins of any host-range class is resistant 40, 42 to superinfection by the same class of virus, whether or not the cell releases any viable progeny virus.

The problem of retroviral host-range interference is specifically illustrated in FIG. 2 in connection with murine cells.

SUBSTITUTE SHEET - 10 -

FIG. 2A shows Eco and Empho retroviral receptors 46, 48 that occur on an uninfected mouse cell 50 having a nucleus 52. Mouse cells 54 that synthesize the Ampho envelope glycoprotein 56, however, lack functional Ampho receptors as shown in FIG. 2B. Conversely, mouse 5 cells 60 that synthesize Eco envelope glycoprotein 62 lack Eco receptors as shown in FIG. 2C.

FIG. 3 illustrates the problem of host-range interference in connection with retroviral packaging cell lines. A psi2 murine cell line 68 contains a nucleus 70, Ampho receptors 72 and cell surface Eco

10 envelope glycoprotein 74. This cell line can potentially release ecotropic host-range virions. Once the retroviral vector 76 is introduced into the cells (for example by DNA mediated transfection), the cells release the "helper-free" virions 78 that contain the vector genomic RNA 80. Because of viral interference (see FIGS. 1 and 2),

15 these virions cannot infect 82 the psi2 packaging cell line from which they were released or any other cell that expresses an ecotropic env gene. The resulting titer of the virion preparation produced by the culture of packaging cells is therefore a low titer, typically about

3 4 10 -10 virions/ml of unconcentrated culture medium. Genetic

20 expression by the packaging cell of the protein encoded by the provirus is also typically low.

The retrovirus formed by any retroviral packaging cell line cannot infect the same packaging cell line. For example, the virus formed using psι^'2 does not affect psi2 cells or any other cell that 5 expresses an ecotropic env gene. However, the virus from psi2 cells can infect PA12 cells, and vice versa.

The retroviral vector can be introduced into the retroviral packaging cell line by DNA mediated transfection. Alternatively, it can be introduced by viral infection. For example, a helper-free

30 virus produced by psi2 cells can be used to infect PA12 cells and vice versa.

If the low titer virion preparation produced by the packaging cell line is used in its unconcentrated form to infect target cells in a culture medium, the genetic expression of the encoded gene will be

35 low. The low titer virion preparation will infect fewer target cells than would be infected with a high titer virion preparation. Multiple infections of individual target cells with many virions will also be relatively infrequent if the low titer preparation is used. - 11 -

Mixed Culture Amplification

The above limits of prior retroviral technology are very serious. The amounts of cloned gene products and of virions that are formed have been typically very small and, therefore, commercially useless. Moreover, even these low yields of the products of interest have required costly, labor-intensive, and time-consuming colony screening procedures. It is possible to overcome some of the aforementioned drawbacks of a low titer virus preparation by concentrating the virions from a large volume of culture medium. Alternatively, one could screen numerous cell clones of the packaging cells to identify the rare few that release a higher titer of virions. It would be desirable, however, to circumvent these serious deficiencies by a novel technology. The present invention solves these problems by using a process that is very simple and relatively inexpensive. Virions that encode cloned genes of interest are formed in relatively large amounts without any colony screening requirement. Moreover, and most importantly, the gene products of interest are formed both in the packaging cells and in the target cells in greatly amplified quantities. These advantages are achieved by introducing a replication defective retroviral expression vector into a mixture of cells from two eukaryotic packaging cell lines that package virions having different host ranges. Retroviral packaging cell lines have already been described that produce virions of differing host-range interference types [Miller et al., Molecular and Cellular Biology 5:431-437 (1985); Mann et al., Cell 33:153-159 (1983); Cone and Mulligan, Proc. Natl. Acad. Sci. U.S.N. 81:6349-6353 (1984)]. For example, the psi cell line described in Mann et al., 1983, is a derivative of murine NIH/3T3 fibroblasts and packages retroviruses into virions with an ecotropic host-range. On the other hand, the PA12 cell line described by Miller et al., 1985, is a derivative of murine NIH/3T3 fibroblasts that packages murine retroviral RNAs into virions that have an a photropic host-range. A nucleic acid that encodes a replication-defective retrovirus (which contains a cloned gene of interest) is introduced into a mixed cell culture of the psι^"2 and PA12 lines to produce virions with different - 12 -

host-range/interference phenotypes. Virions packaged by cells of the psi2 line can then infect PA12 cells, and virions packaged by cells of the PA12 line can infect psi2 cells.

Although numerous methods may be used for introducing the nucleic acid into the culture, a simple method is to simply transfect the nucleic acid into the cells. A major advantage of the present invention is that selection of stably transfected cells is not required. Rather, the cells that transiently incorporate the nucleic acid produce enough virus to initiate the process of amplification shown in FIG. 4.

FIG. 4 shows an Eco packaging cell 90 which contains a nucleus 94, Ampho receptors 96 and cell surface Eco envelope glycoprotein 98. An Ampho packaging cell 100 contains a nucleus 104, Eco receptors 106, and cell surface Ampho envelope glycoprotein 108. A retroviral vector 110 is introduced 112 into a culture that contains a mixture of the Eco and Ampho retroviral packaging cell, lines, which are distinct host-range types. Initiation of the amplification process requires that at least a single cell acquire the retroviral vector 110. The resulting virus then spreads by cross infection 112, 113.

The virus 114, 116 released from each packaging cell type can infect 112, 113 the other cell type. The replication-defective retroviral genome thereby spreads by a process of "back and forth" (ping-pong) infection between the different cell types in the culture. Moreover, the amplification process can theoretically proceed indefinitely because neither packaging cell.type ever becomes resistant to superinfection by virus produced by the other type of cells in the culture. Consequently, each cell in the culture eventually acquires multiple copies of the replication-defective virus and begins to express these viral genes in large quantities. After a period of amplification, the medium from the mixed cell culture contains the replication-defective virus in a substantial concentration. The replication-defective virus can be separated from other components of the culture (such as expressed protein) to produce a high titer virus preparation. These virus preparations have a broad host-range and can be used to infect other cells of interest to the investigator. The amplification in the mixed cultures can be stopped - 12a -

at any time simply by separating the cell types as discrete clones. Each clone of packaging cells expresses the gene product of interest and releases virions of a specific host-range type (i.e., either ecotropic or amphotropic for mixed cultures containing psi2 and PA12 packaging cells).

The following examples will help explain the method.

Example 1 Equal numbers of an ecotropic packaging cell line (2.5 x 10 cells) and an amphotropic packaging cell line (2.5 x 10 cells) were seeded in a 100 mm culture flask. The ecotropic cells were psi2 cells

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as described in Mann et al., 1983, and the amphotropic cells were PA12 cells which were described in Miller et al., 1985. The 10 ml of growth medium was Dulbecco's Modified Eagle Medium (DMEM) containing 10 percent fetal calf serum. The following day, the cells in the culture flask were transfected with 20 μg of plasmid DNA which encoded the defective retroviral genome containing the gene of interest. Plasmids of Friend spleen focus forming virus (SFFV) were used. The plasmids were the wild-type virus plasmid and several viral mutants that were constructed using site-directed mutagenesis methods. Transfection was performed using the calcium phosphate precipitation procedure described in Graham and Van der Eb, Virology 52:456-467 (1973) or Parker and Stark, J. Virol. 31:360-369 (1979).

The transfected mixed cell culture was then allowed to grow for at least two weeks during which the process of cross infection and amplification proceeded. The cultures were fed every three days by provision of fresh medium and were split when they were near or at confluency which typically occurs at 4 to 5 days.

The virions were harvested from the culture media as a virion preparation. To do this, cultures at 50-75 percent confluency had their media exchanged with fresh medium. 18 h to 24 h later, the media were harvested and stored as viral stocks by freezing at -70^βC. No concentration or purification of the virions was performed.

FIGS. 5-7 illustrate some results obtained using the method of Example 1 to amplify expression of a replication-defective retrovirus. These results illustrate that the molecularly cloned retroviruses were amplified in the mixed culture system. The level of gene expression attained in the mixture of packaging cells was much greater than the expression observed with this virus in any other expression system. Moreover, the mixed cell cultures released the cloned viruses in large titers that were used to transfer viral gene expression into other target cells. These target cells then expressed the virus-encoded gene at a high level.

FIG. 5 is an immunoblot (commonly known as a western blot) analysis of cell lysates from mixtures of psi2 and PA12 cells which were used as the packaging cell lines in Example 1. Cell lysates were - 14 -

electrophoresed on SDS polyacrylamide gels and the separated proteins were then transferred to nitrocellulose membranes. These membranes were then allowed to bind antisera directed against the SFFV encoded gene product gp55. A secondary binding with radioactive protein A allowed visualization of the gp55-antibody complexes using autoradiography. Panel A: lane 1, 12 days post transfection; lanes 2 and 3, 6 days post transfection. These results clearly show that amplification of gp55 occurred between 6 and 12 days post transfection. This result also showed that the gp55 glycoprotein was already evident in detectable quatities by 6 days. Panel B: lanes 1 to 5 are five different strains of SFFV that encode gp55s of different sizes. The minor bands in the lanes are nonspecific proteins that cross react with the antiserum employed and that occur in the untransfected mixed cell cultures. Thus, these components are not encoded by the SFFV genome. The immunoblots of FIG. 5 show that mixed cultures, when transfected with molecularly cloned retroviral DNA, express the encoded gene products at very high levels and that the high level expression is the result of amplication of gene expression. FIG. 6 is an immunoblot prepared as described in connection with FIG. 5 but which compares gp55 gene expression using the mixed culture method and standard methods of SFFV expression. Lane 1, mock transfected psi2 and PA12 cells; lane 2, NRK clone 1 cells [Troxler, et al., Proc. Natl. Acad. Sci. U.S.A. 74:4671-4675]; lane 3, SFFV expressed in the mixed culture. A different antiserum was used than that in FIG. 2, and this antiserum reacted relatively strongly with the minor components described in FIG. 2 that are not encoded by the SFFV DNA. This nonspecific component is present in the control lane 1. The two SFFV-specific gp55 components are clearly seen in lanes 2 and 3. The quantity of gp55 is much larger (at least 10-fold) in lane 3 compared with lane 2. This result shows that a cell line isolated using conventional methods of prior technology to obtain expression of SFFV expresses much less gp55 when compared to gp55 expression in the mixed culture system.

FIG. 7 is an immunoblot performed as described in connection with FIG. 5, but which measures SFFV gene expression in target cells infected by the high titer virion preparation produced in Example 1. - 15 -

The five different strains of SFFV which were discussed in connection with FIG. 5 and shown in FIG. 5B, lanes 1-5, were used to infect NIH/3T3 cells. Cell lysates were made 72 hours after infection, and an immunoblot was performed as described in connection with FIG. 5. This result clearly shows that high titer SFFV produced by the mixed cultures can be used to infect target cells causing transfer of the high level SFFV gene expression of gp55.

Table I shows a quantitative titration analysis of a virus recovered from the medium of a mixed culture that had been transfected with the retroviral expression vector pZlPneo-tatlll plasmid [Rosen, ^{et al}-» -• Virol_' 57:379-384 (1986)]. The viruses were harvested from the culture medium in identical conditions according to Example 1 described above. The titers of recovered viruses were determined in NIH/3T3 fibroblasts by standard methods [Cone and Mulligan, Proc. Natl. Acad. Sci. U.S.A. 81:6349-6353 (1984); Southern and Berg, J^ Mol. and Appl. Gen. 1:327-341 (1982)].

Table I

Method of Introducing Titer of Recovered Virus (G418 pZlPneo-tatlll Plasmid into Cells Resistant Colony Forming Units/ml)

Transfection and Subsequent Selection of G418 Resistant Cells 3.6 x 10³

Adding DNA to Mixed Culture 1.4 x 10⁶

The table first shows the results obtained using the methods

5 of prior technology. Transfection of 5 x 10 psi2 cells in a 25

2 cm flask was performed using 10 μg of plasmid DNA by the calcium phosphate precipitation method. The G418 resistant cell colonies were grown as a pooled culture which was then used for the virus harvest.

3 The titer of recovered virus was 3.6 x 10 colony forming units/ml.

In the mixed culture example of Table I, 10 μg of pZIP neotat

III plasmid DNA was transfected using the calcium phosphate

2 precipitation method into a 25 cm flask that had been seeded 24 h

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earlier with 2.5 x 10 psi2 cells and the same number of PA12 cells. The cultured cells were then simply grown for two weeks without any selection and the virus was then harvested. The titer of recovered virus was 1.4 x 10 colony forming units/ml.

The data in Table I illustrate that a relatively high titer of

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virus can be obtained simply by transfecting a molecularly cloned retroviral vector DNA into a mixed culture of cell lines having different host ranges. Elaborate and costly screening methods are not required to perform the method. Alternate Methods for Introducing the Replication-Defective Retroviral Genomes into the Mixed Cell Culture System

Although the simple transient transfection method described i Example 1 for introducing the retrovirus into a mixed cell culture system is easy and effective, other methods are possible. For example the retroviral nucleic acid could be introduced into one of the retroviral packaging cell lines prior to mixing with the other packaging cell line. Moreover, the replication-defective retrovirus could be introduced by infection using a helper-free virion preparatio obtained from any source. Thus, for example, a helper-free virus coul be obtained from psi2 cells using the methods of Miller et al., Somati Cell and Mol. Gen. 12:175-183 (1986); Miller et al., Mol. and Cell. Biol. 5:431-437 (1985); or Miller and Buttimore, Mol. and Cell Biol. 6:2895-2902 (1986). That virus could then be used to infect the mixed cell culture system. It would also be possible to initiate the amplification process by fusing a cell containing a replication-defective retrovirus with a retroviral packaging cell line. The resulting heterokaryon or cell hybrid would release enough of the virus to initiate the amplification. In fact, because the amplification system is so efficient, it should be possible to use it to clonally isolate replication-defective retroviruses that may be expressed in only trace amounts in diseased tissues.

Including a Third Cell Type in the Mixed Cell Cultures

It is possible to include in the mixed cell culture system described above a third cell type that may be uninfected. If this third cell type is susceptible to infection by a replication-defective virus released into the culture medium by one or both of the first two cell lines, the third cell type will also become massively infected and active in expressing the encoded gene. After cocultivation has occurred, the third cell type could be recovered from the mixed culture (see below). It would also be possible to include any number of - 17 -

additional cell types, for example, four or more, in the mixed culture system.

Use of Only One Retroviral Packaging Cell Line

If a retroviral packaging cell line that releases even a low titer of a helper-free replication-defective retrovirus is cocultured with a cell that contains cell surface receptors for the released virus, the latter susceptible cell will eventually become multiply infected. It would therefore begin to synthesize the viral gene product(s) in relatively large quantities. After such an incubation in the mixed culture system, the susceptible cell could be recovered as a pure cell Tine that would stably synthesize a large quantity of the desired viral gene product(s). Recovery of Each Cell Type from the Mixed Cultures

Each cell type that occurs in the mixed cultures can be easily recovered as a pure cell line. Such recovery can be accomplished simply by isolating cell clones from the mixed cultures using standard procedures [Parker, R.C., Methods of Cell Culture, 3rd Edition, 1961, Harper and Rowe, USA]. Alternatively, it is possible to use cell types in the mixed cultures that have dominant selectable markers, e.g., cell surface antigens or dominant genes such as neo [Southern and Berg, J. Molee, and Appl. Gen. 1:327-341 (1982)]. These markers can then be used to isolate and/or to screen the recovered cell clones. The cell lines thereby recovered from the mixed cultures can be individually screened to determine the host-range and titer of the virus that may be released, and to determine the extent of expression of the gene of interest. Thus, for example, it is possible to readily isolate cell clones that release h^'igh-titer preparations of helper-free virus of a defined host-range/interference class. Use of High Titer Virus Preparation Produced by this Mixed Culture Procedure

The method of the present invention facilitates the isolation of cell lines that release high titers of replication-defective viruses that have a defined and potentially broad (e.g., amphotropic) host-range. The viruses produced by these cell lines can be used to infect cells at a high multiplicity, and the infected cells then express the cloned genes in large quantities. The virus preparations - 18 -

can be stably stored (for example by freezing). Therefore, it is possible to produce virus preparations in commercially useful yields and by relatively easy and inexpensive methods. These preparations can be used to cause specific proteins to be formed in target cells of interest to investigators and/or clinicians. Advantages

The present invention has numerous advantages over prior ^' methods for transfer and expression of genes. One of the primary benefits of the present method is that it is simple to perform. Unlike previously used procedures for introducing genes into eukaryotic cells, complex and mutagenic cotransfection is not required, and costly labor-intensive and time-consuming screening procedures are also not necessary. Rather, the cloned gene of interest is added to the cells in a single step and the cloned gene then spreads spontaneously by a massive process of infection within the mixed culture and all cells become converted into high-expresser derivatives.

The level of cloned gene expression in the present method can theoretically become a substantial proportion of total cellular protein synthesis. Using presently available methods and vector systems, the level of gene expression is substantially higher than observed using other methods.

The cells that express the cloned genes remain viable and can easily be isolated as pure healthy cell lines. This aspect of the invention distinguishes the present system from lytic virus syte s (e.g., vaccinia or baculovirus) which cause cell death and, in the case of the baculovirus system, involves expression in insect cells. Consequently, the present method is especially useful for analyzing the physiological function of cloned genes in healthy cells.

The expression of the cloned gene occurs in viable, healthy vertebrate cells. Therefore, secretory proteins are properly processed without the defects that occur when vertebrate proteins are synthesized in the cells of bacteria or lower eukaryotes. Glycoproteins are synthesized and processed correctly. This may be critical to their clinical efficacy. The mixed culture system can be used to produce high titers of replication-defective retroviruses of broad host range that encode - 19 -

cloned genes of interest to investigators and/or clinicians. The present amplification system greatly facilitates such production. Such retrovirus preparations can be used to infect cells of interest to an investigator at a high multiplicity, thereby converting the cells of interest into high expressers. The virus preparations can be stably stored by freezing. Such virus preparations would have broad potential importance for scientific researchers and should therefore be commercially useful. It has not previously been possible to easily, routinely and inexpensively prepare high titer preparations of helper-free retroviruses.

The resulting mixed culture system yields cell lines that express large quantities of cloned genes. These cell lines could be useful for producing gene products of commercial importance. Examples of such gene products include viral vaccine products and glycoprotein hormones. The commercial value of such products will be enhanced by their having been processed and glycosylated in healthy vertebrate cells. The proteins produced by vertebrate cells are properly processed and therefore lack the defects characteristic of proteins produced in oacteria or in lower eukaryotes such as yeast or insects. A very recent review by Gilboa et al., Biotechniques,

4:504-512 (1986) illustrates the need in this field for an efficient process for inserting a gene into a retroviral vector, obtaining recombinant virus, and infecting target cells so that they will express the foreign gene. Many investigators have used other, less satisfactory approaches to overcome the problems that are now solved by the present method.

The mixed culture procedure of the present invention provides a novel and suprisingly useful technology. It is a surprising technology since it was initially doubtful that this procedure would work. "Back and forth" (ping-pong) infection within a mixed cell culture system had, to the inventors' knowledge, not been previously used or proposed to overcome the interference barriers that block virus superinfections. Spread of virus between alternating cell types in a mixed culture is therefore a novel process of infection. For the first time, a method for overcoming the interference barriers that limit infectious transmission and amplification of retroviruses has been - 20 -

achieved.

These advantages were achieved in spite of prior art teachings that could have taught away from the present invention. It has been known for some time that each retroviral packaging cell line synthesizes and secretes into the medium specific env glycoproteins. Thus, psi2 cells shed an ecotropic ervv glycoprotein and PA12 cells shed an amphotropic env glycoprotein. Because cells that synthesize gag, pol and env viral structural constituents bud off virus-like particles into their culture medium regardless of whether the cells contain any packageable retroviral genomic RNA [Levin, et al., J. Virol. 14:152-161 (1974); Shields, et al., Cell 14:601-609 (1978); Jamjoom, et al, ^ Virol. 19:1054-1072 (1976)], it is clear that the packaging cell lines must also release such particles that contain env glycoproteins on their surfaces. It is important to realize that this release occurs whether or not the packaging cells contain any retroviral vector. Furthermore, it is well-known that such shed env glycoproteins and virus-like particles can competitively bind to and saturate the cellular receptors that mediate retroviral uptake [Moldow, et al., Virology 97:448-453 (1979); DeLarco and Todaro, Cell 8:365-371 (1976); Steck and Rubin, Virology 29:628-641 (1966); Weiss, R. in RNA Tumor Viruses, R. Weiss, N. Teich, H. Varmus and J. Coffin (eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982, pp. 236-247; Tozawa, et al., Virology 40:530-537 (1970); Robinson, et al., J. Virol. 36:291-294 (1980); Fowler, et al., J. Virol. 24:729-735 (1977); Johnson and Rosner, J. Virol. 58:900-908 (1986); Bishayee et al., Archives Biochem. Biophys. 189:161-171 (1978); Altrock, et al., Vi ology 109:257-266 (1981)]. This competitive blocking of infections is called "early interference" [Steck, et al. (1966); Weiss, R. in RNA Tumor Viruses, pp. 236-247 (1982)]. When the present invention was initiated, it seemed certain that such early interference would occur and that it could potentially prevent or severely inhibit the ping-pong mode of virus transmission that would be required for successful retrovirus vector amplification in a mixed culture system. In addition, the ping-pong process for retrovirus amplification in a mixed cell culture involves multiple cycles of virus infection into cells, followed by release of the transmitted virus. It has been accepted - 21 -

that retrovirus replication is an error-prone process so that mutations would propagate and increase in proportion throughout the amplification process (Joyner and Bernstein, Mol. and Cell. Biol. 3:2191-2202 [1983]; Sorge, et al., Proc. Natl. Acad. Sci. U.S.A. 84:906-909 [1987]). This effect would reduce the efficacy of the mixed culture system. However, our results have indicated that this effect does not prevent successful amplification of functionally active and intact test genes.

Example 2 The same procedure could be used as in Example 1, except PA317 cell lines could be substituted for PA12. Cells could also be transfected with 10 μg of plasmid DNA instead of the 20 μg of Example 1.

Example 3 The harvested virions from Example 1 were used to infect cells of a target cell line. The target cells were NIH/3T3 mouse fibroblasts which were infected by exposing them to complete medium containing the harvested virions supplemented with 8 μg/ml polybrene. The NIH/3T3 cells were infected for two hours at 37^βC. The medium was then replaced with normal growth (DMEM plus 10 percent fetal calf serum) medium and cells were allowed to grow for two to three days before analysis of the expression of the viral-encoded genes.

Example 4 The procedure of Example 1 could be modified such that the mixed cell culture would include cells from the packaging cell lines and the cells of a target cell line would be added after amplification. In this procedure, a target cell line is chosen which is susceptible to infection by the virus released by the packaging cells. An example would be to use as a target cell NIH/3T3 mouse fibroblasts which express receptors for the viruses produced by both PA12 and psi2 cells. By cocultivating the NIH/3T3 cells with the PA12 plus psi2 virus-producing cell lines, continual infection of the target cells would occur, resulting in amplified expression of the virion-encoded gene(s) in the target NIH/3T3 cells.

Example 5 Instead of harvesting virions and using them to infect target cells, as in the procedure of Example 3, whole animals could be infected by injecting high titer virions produced by cells from a mixed - 22 -

culture. An injection protocol into whole animals using prior art technology to obtain virions was described by Bestwick et al., J. Virol 56:660-664 (1985) and Wolff and Roscetti, Science 228: 1549-1552 (1985). The term "whole animal" means any vertebrate, including humans, whose cells contain receptors for the virions produced by the mixed culture system. The result would be to cause in vivo expression of the cloned gene(s) encoded by the retroviral vector.

Example 6 Another method of causing in vivo expression of the cloned gene of interest would be to transplant multiply-infected high-expressor target cells of Examples 3 or 4 into a whole animal. This procedure would be accompl shed by using target cells that could be transplanted into the animal without causing immunological rejection. Such target cells ^"could be obtained by prior biopsy of the host animal itself.

Example 7 The procedure of Example 1 could be used to produce a foreign gene product in large amounts. An example of a gene that could be used in this process is human growth hormone. By constructing a defective retroviral vector capable of expressing human growth hormone, the mixed cell process could be used to produce cells expressing amplified levels of human growth hormone.

While we have shown and described preferred embodiments of our invention, it will be apparent to those skilled in the art that changes may be made without departing from our invention in its broader aspects. The appended claims are therefore to cover changes and modifications as follow in the true spirit and scope of our invention.

Claims

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WE CLAIM:

I. A process for amplifying expression and transmission of cloned genes in eukaryotic cell cultures, comprising: introducing a replication defective retroviral expression vector for expressing a cloned gene into a mixed culture of cells from first and second eukaryotic packaging cell lines that package virions with different host ranges; and maintaining the mixed culture under conditions which allow the production of virions and increased infection of the cells.

2. The process of claim 1 in which the vector is introduced into the mixed culture by transfection using a nucleic acid.

3. The process of claim 1 in which the vector is introduced into the mixed culture by infection with a helper-free retrovirion.

4. -The process of claim 1 in which the vector is introduced into the mixed culture by fusion of a cell that contains the vector with a cell in the mixed culture of packaging cells.

5. The process of claim 1 which further includes the step of separating the mixed culture of cells into separate pure cell lines, ._ after the production of virions, to terminate infectious amplification in the mixed culture.

6. The process of claim 5 which further includes the step of harvesting, from at least one of the separated cell lines, virions that are released into the culture medium.

7= The process of claim 5 which further includes the step of harvesting, from at least one of the separated cell lines, gene products that are encoded by the retroviral expression vector.

8. The process of claim 1 which further includes the step of harvesting, from the mixed culture, gene products encoded by the retroviral expression vector.

9. The process of claim 1 wherein the mixed culture includes more than the two cell lines.

10. The process of claim 1 further including the step of harvesting virus produced by the mixed culture.

II. ^'The process of claim 10 further including the step of introducing the harvested virus into the cells of a whole animal. - 24 -

12. The process of claim 10 further including the step of infecting the cells of a target cell line with the harvested virus.

13. The process of claim 12 further including the step of isolating, from infected cells of the target cell line, gene products encoded by the retroviral expression vector.

14. The process of claim 12 further including the step of introducing into a whole animal, target cells that actively express a gene product of interest.

15. The process of claim 10 further including the step of - storing the harvested virus in conditions suitable for preserving its infectious ability.

16. The process of claim 14 wherein the step of storing includes frozen storage.

17. The process of claim 1 further including the step of introducing into a whole animal, cells from the mixture that actively express a gene product of interest.

18. The process of claim 1 wherein the step of introducing the expression vector into the mixed culture includes the step of first introducing the vector into cells of the first packaging cell line, then mixing cells of the first packaging cell line with cells of the second packaging cell line.

19. A process for producing gene products, including the steps of: introducing a replication defective retrovirus into cells of a eukaryotic retroviral packaging cell line and allowing the cells to produce and release virions; coculturing the cells of the packaging cell line with eukaryotic cells which have surface receptors for the released virions and thus are susceptible to infection.

20. The process of claim 19 wherein the process further includes the step of allowing cells of the susceptible cell line to become multiply infected, then recovering cells of the susceptible cell line to provide a pure cell line.

21. The pure cell line of claim 20.

22. A process of forming a desired protein encoded by a gene of interest, the process including the steps of: - 25 -

introducing a gene of interest that has been cloned into a retroviral expression vector into cells of a first eukaryotic packaging cell line; coculturing the cells of the first packaging cell line with ceTls of a second eukaryotic packaging cell line, the first and second packaging cell lines having different host ranges; allowing the cells of the first and second cell lines to produce virions, each packaging cell line having receptors for the virions released by the other packaging cell line; ° infecting cells of a target cell line with the virions to produce a product of the gene of interest by expression of the gene; and isolating the product.

23. A process for amplifying expression and transmission of cloned genes in eukaryotic cell cultures, comprising: 5 introducing a replication defective retroviral expression vector for expressing a cloned gene into a mixture of cells, the mixture including a eukaryotic packaging cell and a target cell which is susceptible to infection by the virus released by the packaging cell.

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