Modified cell lines for increasing lentiviral titers
Inventors
Spayd, Katie Jansen • Karpilow, Jon • Wakefield, John K.
Assignees
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Publication Number
US-9284552-B2
Publication Date
2016-03-15
Expiration Date
Abstract
The present disclosure provides a method of modifying cells in order to enhance lentiviral titers, cell lines that are modified and modifying reagents. By mediating individual genes and combination thereof, lentiviral titers may be increased.
Core Innovation
The invention enhances lentiviral titers by modulating expression of a plurality of host genes in a packaging cell. The modulation comprises suppressing expression of Tetherin and at least one additional modulation selected from suppressing expression of at least one of Dicer and DGCR8, and increasing expression of at least one of TSG101 and Anx2.
The problem being solved is that lentiviral titers are enhanced by modifying packaging cells through host gene modulation, including identifying inhibitory host genes and facilitator genes that affect lentiviral titers. Inhibitory host genes identified include Dicer, Drosha, DGCR8, Hrs, and Tetherin, and facilitator genes identified include Annexin2 (Anx2) and Tsg101.
The described results indicate that RNAi-pathway knockdowns of Drosha, Dicer, and DGCR8 increase lentiviral titers, and DGCR8 knockdown can boost titers about two-fold or more depending on siRNA. Tetherin knockdown significantly increases titers, and overexpression of Tsg101 and Annexin2 is described as increasing lentiviral titers.
The document also states predicted advantages of combining multiple gene modulations and generating stable modified cell lines for high-titer production.
Claims Coverage
The independent claim identified in the partial content covers a method of enhancing lentiviral titers using modulation of multiple host genes in a packaging cell, comprising one required modulation and at least one additional modulation. The claim set further includes dependent limitations that specify particular modulation forms and quantitative thresholds in some dependents.
Suppressing tetherin in a packaging cell
A method of enhancing lentiviral titers comprising modulating expression of a plurality of host genes in a packaging cell, wherein the modulation comprises suppressing expression of Tetherin.
Suppressing dicer and/or dgcr8 or increasing tsg101 and/or anx2
The method further includes at least one additional modulation selected from suppressing expression of at least one of Dicer and DGCR8, and increasing expression of at least one of TSG101 and Anx2.
Two shRNA constructs targeting tetherin and dgcr8
The method further modulates by expressing two shRNAs in a packaging cell, wherein the first shRNA inhibits Tetherin and the second shRNA inhibits DGCR8.
Overexpressing tsg101
The method further comprises overexpressing TSG101 by at least 30%.
Suppressing tetherin by a quantitative range
The method further suppresses Tetherin by 30% to 40%.
Inhibiting tetherin with shRNA and overexpressing anx2
The method further modulates by expressing shRNA in a packaging cell to inhibit Tetherin while causing overexpression of Anx2.
Gene participation in an rna i pathway
The method includes a limitation that at least one gene participates in an RNAi pathway.
Overall claim coverage is centered on enhancing lentiviral titers by suppressing Tetherin in a packaging cell and performing at least one additional modulation that can include suppressing Dicer and DGCR8 and/or increasing TSG101 and Anx2. The dependents further specify modulation via shRNA constructs, quantitative overexpression and suppression thresholds, and that at least one targeted gene participates in an RNAi pathway.
Stated Advantages
Enhances lentiviral titers.
Combining multiple gene modulations is predicted to be advantageous for high-titer production.
Generating stable modified cell lines is predicted to support high-titer production.
Documented Applications
Use of modified packaging cells in lentiviral transfer/packaging contexts to improve lentiviral titers.
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