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US 4,861,719 AGranted
DNA constructs for retrovirus packaging cell lines
AssigneeFred Hutchinson Cancer Research Center
InventorA. Dusty Miller
Life of this patent
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AssigneeFred Hutchinson Cancer Research Center
InventorA. Dusty Miller
Estimating the term…
DNA constructs consisting essentially of the promoter, gag, pol, and env sequences of a helper virus useful for making retrovirus packaging cell lines that do not yield helper virus and do not transfer the packaging function. Such DNA molecules are constructed by deleting from the genome of a replication-competent retrovirus all cis-acting elements except for the tRNA binding site. Specifically, deletion is made of the packaging signal, the site for initiation of second strand DNA synthesis, the site required for translation of reverse transcriptase during first strand DNA synthesis, and the provirus integration signal. DNA construct pPAM3 (ATCC No. 40234) is a representative embodiment. A cell line containing such an altered viral genome does not transmit this virus or transfer the packaging signal, but will transmit high titers of other viral RNAs containing the proper cis-acting elements, including retroviral vectors designed to carry foreign genes. Cell line PA317 (ATCC No. CLR 9078) is a representative embodiment.
An improved DNA construct useful for making retrovirus packaging cell lines comprising an amphotropic retroviral gene sequence derived from murine leukemia virus encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and characterized by its ability, when transferred into susceptible host cells, to produce virion proteins capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus, which vector contains a heterologous gene, said retroviral gene sequence lacking a packaging signal, a site for translocation of reverse transcriptase during first strand DNA synthesis, a site for initiation of second strand DNA synthesis and a proviral integration signal.
A DNA construct having the characteristics of pPAM3 (ATCC No. 40234).
A process for constructing an improved DNA construct useful for making retrovirus packaging cell lines, comprising deleting from the genome of a replication-competent amphotropic retrovirus derived from murine leukemia virus, the packaging signal, the site of initiation of second strand DNA synthesis, the site required for translocation of reverse transcriptase during first strand DNA synthesis, and the provirus integration signal.
A process for constructing a retrovirus packaging cell line comprising the steps of:
A packaging cell line comprising a DNA construct having an amphotropic retroviral gene sequence derived from murine leukemia virus encoding in trans all virion proteins required for packaging a replication-incompetent retroviral vector and lacking a packaging signal, a site for translocation of reverse transcriptase during first strand DNA synthesis, a site for initiation of second strand DNA synthesis and a proviral integration signal, and packaging cell line being capable of packaging said replication-incompetent retroviral vector at high titer, without the production of replication-competent helper virus.
A cell line having the characteristics of PA317 (ATCC No. CRL 9078).
Dependencies are inferred from the claim text.