Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2001-09-24
2004-09-21
Wehbe′, Anne M. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S325000, C435S320100, C435S455000, C514S04400A, C424S093100, C424S093200, C800S013000, C800S018000
Reexamination Certificate
active
06794188
ABSTRACT:
FIELD OF INVENTION
This invention relates to the fields of genetic engineering and gene transfer. More specifically, the invention relates to recombinant retrovirus vectors derived from avian sarcoma leukosis viruses (ASLVs) having an expanded host range. In particular, this invention relates to ASLV recombinant retrovirus vectors wherein a viral env gene derived from a virus capable of infecting both mammalian and avian cells is substituted for the ASLV env gene, allowing the vectors to efficiently infect a wide range of host cells, including mammalian and particularly human cells, in high titers. Additionally, this invention encompasses therapeutic applications employing these vectors.
BACKGROUND OF INVENTION
Retroviral vectors carrying and expressing nucleic acid sequences of interest are powerful tools for the transfer of genes into a broad range of mammalian cells and into animals, including humans. Indeed, retroviruses offer substantial advantages for use as vectors carrying and expressing desired nucleic acid sequences in both cultured cells and intact animals. (Weiss et al,
RNA Tumor Viruses
(1982)).
First, the retrovirus life cycle lends itself to the efficient transfer of genes into host cells. The infectious retroviral agent is called a viral particle or a virion. Virions consist of a capsid containing the viral genome and any inserted nucleic acid sequences and an envelope made up of glycoproteins. The envelope glycoproteins on the surface of the virion recognize receptors on the host cell that mediate entry of the RNA retroviral genome into the host cell. Once inside the host cell, a double stranded DNA copy of the virion RNA genome and any inserted nucleic acid sequences of interest is made by a viral enzyme, reverse transcriptase. This DNA copy integrates into the host genome at a precise point on the viral DNA molecule and at random, or nearly random sites on host chromosonal DNA. The integrated viral DNA copy is called a provirus. Since a DNA copy of the viral genome integrates into the host genome, the progeny of a single infected host cell are all infected, and the provirus is located in the same place in the genome of each of the progeny cells.
Second, in completing their replicative process, retroviruses usually do not lyse the host cell. Thus, the retroviruses constitute an efficient mechanism for the introduction and high level expression of genes in living host cells.
Third, retroviral genomes are small, making it relatively easy to manipulate a cloned DNA copy of the genome. Moreover, the viruses are efficient; in culture, essentially all of the cells can be infected.
The ability of the retroviral replication machinery to introduce genetic information into the genome of the target cell provided the inspiration for the development of recombinant retrovirus vectors containing a nucleic acid sequence of interest as a vehicle for the stable transfer of genes. Moreover, recombinant retroviral vectors have been used in a number of applications in addition to the expression of genes of interest, including insertional mutagenesis, cell lineage studies and the creation of transgenic animals.
A desirable property useful for the retroviral vector is the ability to replicate in certain easily manipulated host cells, (e.g., avian cells) allowing rapid replication in these cells without aid of a helper or packaging cell line. This permits generation of high titer virus stocks by simply passaging transfected cells and allowing the virus to spread.
Another useful property for a retroviral vector is the ability to infect a wide range of host cells, including mammalian, and particularly human, cells in high titers. Preferably, the retroviral vector is unable to replicate in mammalian cells. Thus, once the vector enters the mammalian host cell, it becomes a stable provirus, integrated in the host cell genome and incapable of further rounds of infection in either the present or subsequent generations.
A number of retroviral vector systems have been described, including systems based on both mammalian (murine leukemia virus, Cepko, et al., (1984)
Cell
37:1053-1062, Cone and Mulligan, (1984)
PNAS
(U.S.A.) 81:6349-6353; mouse mammary tumor virus, Salmons et al., (1984)
Biochem. Biophys. Res. Commun.
159:1191-1198; gibbon ape leukemia virus, Miller et al. (1991)
J. Virology,
65:2220-2224; human immunodeficiency virus, Buchschacher and Panganiban, (1992)
J. Virology
66:2731-2739, Page et al., (1990)
J. Virologv
64:5270-5276) Shimada et al., (1991)
J. Clin. Invest.
88:1043-1047); and avian retroviruses (Boerkoel et al., (1993)
Virology
195:669-679, Cosset et al., (1990)
J. Virology
64:1070-1078, Greenhouse et al., (1988)
J. Virology
62:4809-4812, Hughes et al., (1986)
Poult. Sci.
65:1459-1467, Petropoulos and Hughes, (1991)
J. Virology
65:3728-3737, Valsessia et al., (1992)
J. Virology
66:5671-5676). However, none of these vector systems combines all of the above features. Indeed, each of the available retroviral vectors suffers from certain disadvantages.
For example, one of the most widely used retroviral vectors is a replication-defective derivative of Moloney murine leukemia virus (MLV). The main advantage of MLV is that it has a wide host range and can infect mammalian host cells, including human cells. However, the vectors derived from this virus are replication-defective. MLV vectors contain all of the cis-active elements necessary for viral replication, but lack the genes for the viral structural proteins. These proteins must be provided in trans by a helper or packaging cell line.
MLV and other replication-defective vectors have two major disadvantages. First, the titers of recombinant retrovirus produced by a helper or packaging cell line are not always sufficient for some applications, for example, for in vivo gene transfer experiments or gene therapy. (See, e.g. Hopkins, (1993)
PNAS
(U.S.A.) 90:8759-8760). Second, recombination events between the helper or packaging cell line genome and the replication-defective vector can occur and can result in the generation of wild-type virus. (Ott et al., (1994)
Hum. Gene Ther.
5:567-575). Contamination of the recombinant retroviral vector stock with replication-competent MLV can interfere with gene transfer and present potentially serious problems if the vector is used for gene therapy. For example, leukemias and lymphomas were induced in primates infected by the wild-type MLV contaminating retroviral vector stocks. (Donahue et al., (1992)
J. Exp. Med.
176:1125-1135; Vanin et al., (1994)
J. Virology
68:4241-4250). Finally, in order to use a helper or packaging cell line, a selectable marker must be introduced into the retroviral vector. However, with a helper-independent system there is no need to introduce a selectable marker into the vector, since any sequence present in the vector will be carried along passively during replicants.
Other frequently used retroviral vectors are derived from avian sarcoma leukosis viruses (ASLVs), particularly the Rous sarcoma virus (RSV). (Hughes and Kosik (1984) Virology 136:89-99; Hughes et al., (1987)
J. Virology
61:3004-3012). RSV is the only known replication-competent retrovirus carrying an additional gene, oncogene v-src, which is dispensable for viral replication. This oncogene can be deleted from the RSV derived vector and replaced with a gene or genes of interest without affecting the ability of the virus to replicate. For example, retroviral vectors derived from RSV in which the v-src sequences present in the parental RSV have been replaced with a unique restriction site, Cla I, which can be used to insert the gene or genes of interest have previously been described. These vectors are designated the RCAS series. (Hughes et al., (1987)
J. Virology
61:3004-3102). The stability of these vectors was improved by removal of the direct repeat upstream of the src region. (Hughes et al., (1987)
J. Virology
61:3004-3102). The construction and advantages of these vectors are described in Petropoulos and Hughes (1991)
J. Virology
65:3728-3737. (See a
Barsov Eugene
Hughes Stephen H.
Leydig , Voit & Mayer, Ltd.
The United States of America as represented by the Department of
Wehbe′ Anne M.
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