Retroviruses and vectors derived from them require an envelope protein in order to transduce efficiently a target cell. The envelope protein is expressed in the cell producing the virus or vector and becomes incorporated into the virus or vector particles. Retrovirus particles are composed of a proteinaceous core derived from the gag gene that encases the viral RNA. The core is then encased in a portion of cell membrane that contains an envelope protein derived from the viral env gene.
The envelope protein is produced as a precursor, which is processed into two or three units. These are the surface protein (SU) which is completely external to the envelope, the transmembrane protein (TM) which interacts with the SU and contains a membrane spanning region and a cytoplasmic tail (Coffin 1992 In The Retroviridae, Pleum Press, ed Levy). In some retroviruses a small peptide is removed from the TM.
In order to act as an effective envelope protein, capable of binding to a target cell surface and mediating viral entry, the envelope protein has to interact in a precise manner with the appropriate receptor or receptors on the target cell. This must occur in such a way as to result in intemalisation of the viral particle in an appropriate manner to deliver the genome to the correct compartment of the cell to allow a productive infection to occur.
There have been many attempts to use the envelope protein derived from one virus to package a different virus, this is known as pseudotyping. The efficiency of pseudotyping is highly variable and appears to be strongly influenced by interactions between the cytoplasmic tail of the envelope and the core proteins of the viral particle. The process by which envelope proteins are recruited into budding virions is poorly understood, although it is known that the process is in someway ordered as most cellular proteins are excluded from retroviral particles (Hunter 1994 Semin. Virol. 5:71-83). In some retroviruses budding may occur in the absence of envelope proteins indicating that the env is not necessary for this process but conversely the core can influence the efficiency of envelope incorporation into the particle (Einfeld 1996 Curr. Top. Microbiol. Immunol. 214:133-176; Kräusslich and Welker 1996 Curr. Top. Microbiol. Immunol. 214:25-63).
There is evidence for a precise molecular interaction between a cytoplasmic domain of the envelope protein and the viral core in some retroviruses. By way of example, Januszeski et al (1997 J. Virol. 71: 3613-3619) have shown that minor deletions or substitutions in the cytoplasmic tail of the murine leukemia virus (MLV) envelope protein strongly inhibit incorporation of the envelope protein into viral particles. In the case of HIV-1, Cosson (1996 EMBO J. 15:5783-5788) has shown a direct interaction between the matrix protein of HIV-1 and the cytoplasmic domain of its envelope protein. This interaction between the matrix and envelope protein plays a key role in the incorporation of the envelope protein into budding HIV-1 virions. This is shown by the fact that visna virus can only be efficiently pseudotyped with HIV-1 envelope protein if the amino terminus of the matrix domain of the visna virus gag polyprotein is replaced by the equivalent HIV-1 matrix domain (Dorfman et al, 1994 J. Virol. 68:1689-1696).
However the situation is complex, since truncation of the HIV-1 envelope protein is required for efficient pseudotyping of Molony murine leukemia virus (Mammano et al, 1997 J. Virol. 71:3341-3345), whilst truncation of the human foamy virus envelope protein reduced its ability to pseudotype murine leukemia virus (Lindemann et al, 1997 J. Virol. 71:4815-4820). There is also an environmental component to the interaction between the core of a retrovirus and the cytoplasmic tail of its envelope protein. Prolonged passage of EIAV in some cell lines results in a truncation of the glycoprotein, suggesting that host cell factors can select for a virus on the basis of the C-terminal domain of the envelope protein (Rice et al, 1990 J. Virol. 1990 64: 3770-3778).
These studies and those of many other workers indicate that it is not possible to predict that even closely related retroviruses may be able to pseudotype each other. Further more, if a given envelope protein fails to pseudotype a particular virus, it is not possible to predict the molecular changes that would confer the ability to pseudotype. Pseudotyping has met with some success, but is clearly constrained by the need for compatibility between the virus components and the heterologous envelope protein.
In the construction of retroviral vectors it is desirable to engineer vectors with different target cell specificities to the native virus, to enable the delivery of genetic material to an expanded or altered range of cell types. One manner in which to achieve this is by engineering the virus envelope protein to alter its specificity. Another approach is to introduce a heterologous envelope protein into the vector to replace or add to the native envelope protein of the virus.
The MLV envelope protein is capable of pseudotyping a variety of different retroviruses. MLV envelope protein from an amphotropic virus allows transduction of a broad range of cell types including human cells.
The envelope glycoprotein (G) of Vesicular stomatitis virus (VSV), a rhabdovirus, is another envelope protein that has been shown to be capable of pseudotyping certain retroviruses. Its ability to pseudotype MoMLV-based retroviral vectors in the absence of any retroviral envelope proteins was first shown by Emi et al (1991 Journal of Virology 65:1202-1207). WO94/294440 teaches that retroviral vectors may be successfully pseudotyped with VSV-G. These pseudotyped VSV-G vectors may be used to transduce a wide range of mammalian cells. Even more recently, Abe et al (J Virol 1998 72(8) 6356-6361) teach that non-infectious retroviral particles can be made infectious by the addition of VSV-G.
Burns et al (1993 Proc. Natl. Acad. Sci. USA 90: 8033-7) successfully pseudotyped the retrovirus MLV with VSV-G and this resulted in a vector having an altered host range compared to MLV in its native form. VSV-G pseudotyped vectors have been shown to infect not only mammalian cells, but also cell lines derived from fish, reptiles and insects (Burns et al 1993 ibid). They have also been shown to be more efficient than traditional amphotropic envelopes for a variety of cell lines (Yee et al, 1994 Proc. Natl. Acad. Sci. USA 91: 9564-9568, Lin, Emi et al, 1991 Journal of Virology 65:1202-1207). VSV-G protein can be used to pseudotype certain retroviruses because its cytoplasmic tail is capable of interacting with the retroviral cores.
The provision of a non-retroviral pseudotyping envelope such as VSV-G protein gives the advantage that vector particles can be concentrated to a high titre without loss of infectivity (Akkina et al, 1996 J. Virol. 70: 2581-5). Retrovirus envelope proteins are apparently unable to withstand the shearing forces during ultracentrifugation, probably because they consist of two non-covalently linked subunits. The interaction between the subunits may be disrupted by the centrifugation. In comparison the VSV glycoprotein is composed of a single unit. VSV-G protein pseudotyping can therefore offer potential advantages.
However, there are certain disadvantages involved in using producer cell lines to manufacture retrovirus vectors pseudotyped with VSV-G. The first is the difficulty in producing stable cell lines that express VSV-G; the second is the limited life spans of such cell lines.
A number of workers have reported that constitutive high-level expression of VSV-G is toxic to most mammalian cells (eg Emi et al, 1991 Journal of Virology 65:1202-1207, Yee et al, 1994 Proc. Natl. Acad. Sci. USA 91: 9564-9568). A variety of approaches have been used to solve these problems. By way of example, Yee et al (1994 Proc. Natl. Acad. Sci. USA 91: 9564-9568) developed a scheme for producing VSV-G pseudotypes by first producing 293 cell lines that constitutively express gag-pol proteins and contain a retroviral genome. These cell lines were then transfected with a plasmid containing the VSV-G gene downstream of a human cytomegalovirus immediate early promoter followed by the splicing and polyadenylation signals derived from the rabbit β-globin gene. Maximal production of transducing particles was obtained between 48 and 72 hours after transfection.
WO96/35454 teaches that a tetracycline responsive promoter may be used in combination with a nucleotide sequence enocoding vesicular stomatitis virus (VSV-G) to derive a retroviral packaging cell line that inducibly expresses the VSV-G protein, at levels sufficient to support high level virus production, but without the toxic effects of constitutive expression of VSV-G. Ory et al (1996 Proc. Natl. Acad. Sci. USA 93:11400-11406) used the tetR/VP 16 transactivator and tetracycline responsive operator (tet0) minimal promoter system for inducible, tetracycline-regulatable expression of VSV-G in the production of packaging 293 cell lines. Yang et al (1995 Human Gene Therapy 6:1203-1213) used a similar strategy linking seven copies of the tet0 to a minimal HCMV promoter to construct packaging lines derived from NIH-3T3 cells. Chen et al (1996 Proc. Natl. Acad. Sci. USA 93: 10057-10062) modified the tetracycline-inducible system (Gossen & Bujard, 1992 Proc. Natl. Acad. Sci. USA 89: 5547-5551) by fusing the ligand binding domain of the estrogen receptor to the carboxy terminus of a tetracycline-regulated transactivator. Using this system, they constructed cell lines that expressed VSV-G in the absence of tetracycline. VSV-G expression could be induced by β-estradiol regardless of whether the cells were grown with or without tetracycline. However, induction of VSV-G expression was higher when tetracycline was not present. This allowed the construction of stable packaging cell lines that produced transducing viral particles.
Yoshida et al (1997) developed an adenovirus system to produce MoMLV vectors pseudotyped with VSV-G. First a cell line was produced containing a genome plasmid. Secondly this cell line was infected with three different adenoviruses, one encoding the gag-pol gene of MoMLV under the control of the tetracycline transactivator, the second encoding VSV-G under the control of the tetracycline transactivator and the third encoding a nuclear localising transactivator. Transducing particles could be harvested from the resultant cells for a limited time period. Other researchers developing systems to study the export and processing of VSV glycoprotein mutants have used vaccinia virus systems in which the glycoprotein gene was cloned downstream of a bacteriophage T7 promoter. Co-infection of cells with the glycoprotein encoding vaccinia and a vaccinia virus expressing T7 polymerase resulted in a high level of expression of the VSV-G protein (Lefkowitz et al, 1990, Virology 178;373-383).
Arai et al (1998 J. Virol. 72:1115-1121) commented on the fact that the cell lines, in which the expression of VSV-G was controlled by the tetracycline-inducible system, produced low titres of transducing particles in the presence of tetracycline when VSV-G expression should be repressed. This leaky virus production by these packaging cell lines before induction could cause both virus re-entry into the cell culture and accumulation of the vector DNA in the chromosomes during the process of selection and subsequent passages of the packaging cell lines harbouring the virus vector.
Arai et al (1998) reported the development of packaging cell lines in which a completely silent gene for the VSV glycoprotein was present to negate the above problem. This was achieved using a system in which a cassette was produced which encoded the CAG (the chicken β-actin gene promoter connected with the cytomegalovirus immediate-early promoter) followed by the 5′ loxP sequence followed by the neo gene with an associated poly A signal followed by the 3′ loxP sequence followed by the coding sequence for VSV-G and an associated poly A signal. When transfected into cells only the neo gene product is produced. If an adenovirus encoding the Cre recombinase is then introduced into the cell the neo sequence is removed by recombination and the VSV-G gene is expressed from the CAG promoter.
None of these approaches actually solve the problem associated with genome re-entry into cells once VSV-G expression has been initiated. Although the cell lines produced by Arai et al (1998) will be stable until infected with the Cre recombinase encoding adenoviruses, their results indicated that virus production dropped significantly 5 days after adenovirus infection allowing a limited number of harvests from each batch of producer cells.
Chen et al (1996 Proc. Natl. Acad. Sci. USA 93: 10057-10062) produced two tetracycline/β-estradiol-inducible cell lines which expressed VSV-G. The numbers of transducing particles produced every 48 hours increased over a sixteen days period after induction by either the removal of tetracycline from the medium or by the addition of β-estradiol in the absence of tetracycline in the cell line that produced the lowest level of VSV-G. However in the cell line that produced larger amounts of VSV-G, although an increase in titre was observed in the absence of tetracycline, a rapid fall in the number of transducing particles produced was found when high levels of VSV-G were produced upon β-estradiol induction in the absence of tetracycline. This fall was attributed to the toxic effects of high levels of VSV-G expression. Additional examples of proteins toxic to cells when expressed at high levels are the gag/pol proteins of FIV and HIV.
The present invention seeks to provide an improved method for regulating expression of toxic proteins required for vector production but which are inhibitory to cell growth.