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Modified Cell Lines for Increasing Lentiviral Titers

Inactive Publication Date: 2012-04-05
DHARMACON INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0004]The present invention provides modified packaging cell lines and methods for efficient production of lentiviral particles. Specifically, in some embodiments the cells have been altered in such a way as to knockout or knockdown one or more host encoded genes that negatively affect lentiviral vector particle formation in the packaging cell line. Alternatively or in addition to the aforementioned alterations, the cells have been modified to over-express one or more host encoded genes that facilitate viral particle formation and release. Host genes that can

Problems solved by technology

The inventors have recognized that the current set of reagents and protocols is highly inefficient and that new methods are needed to develop sufficient quantities of viral particles.

Method used

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  • Modified Cell Lines for Increasing Lentiviral Titers
  • Modified Cell Lines for Increasing Lentiviral Titers
  • Modified Cell Lines for Increasing Lentiviral Titers

Examples

Experimental program
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Effect test

example i

Effects of Knockdown of Genes Associated with the RNAi Pathway on Lentiviral Titer

[0030]To determine the effects of the RNAi pathway on lentiviral titer, HEK 293T cells (ATCC) were first seeded (6×105 cells / well, 6 well plate) and cultured for 18-24 hours. Subsequently, a pool of siRNAs (SMARTpools, Thermo Fisher Scientific, Dharmacon Products) targeting 1) Dicer (cat. no. M-003483-00-0005), 2) Drosha (cat. no. M-016996-02-0005), or 3) DGCR8 (cat. no. M-015713-01-0005) were transfected (25 nM) into the cells by lipid mediated transfection (DharmaFECT 1, Thermo Fisher Scientific, Dharmacon Products) using manufacturer's recommended procedures. The pools of siRNA were duplexes that contained sense strands that contained the sequences identified in Table I.

[0031]Twenty-four hours post-transfection (Day 3) cells were co-transfected with a pool of plasmids that included a lentiviral transfer vector (6 micrograms / well) and accessory packaging vectors (TransLenti Packaging Mix, Thermo Fish...

example ii

Effects of Knockdown of Hrs and Tetherin on Lentiviral Titers

[0035]Using the original protocol, the researchers investigated the effects that siRNA-mediated knockdown (KD) of Hrs and Tetherin (Thermo Fisher Scientific, Dharmacon Products, cat. no. M-011817-00-0005 (Tetherin) and M-016835-00-0005 (Hrs)) would have on lentiviral titers. As shown in FIG. 5, KD of Hrs provided only modest increases in viral titers (<1.5× effects). In contrast, KD of Tetherin induced significant increases in viral titer (greater than two fold, p<0.05).

example iii

Effects of Over-Expression of Annexin2 and Tsg101 on Lentiviral Titers

[0036]To test the effects that over-expression of Annexin2 has on lentiviral titers, lentiviral particles capable of expressing the Annexin2 or Tsg101 open reading frame (ORF) were transduced into HEK293T cells at an MOI of either 0.3 or 3.0 (LentiORF, Thermo Fisher Scientific, Open Biosystems Products cat. no. PLOHS—100003638 (Annexin 2) and PLOHS—100005422 (Tsg101)). Subsequently, cultures were grown in the presence of blasticidin (10 ug / ml, 2 week) to select for stable integrants. Cells were then expanded for 1 week in the absence of blasticidin, seeded at a density of 1.2×106 cells per well in a 6 well plate, and then transfected with a transfer vector / Trans-Lenti packaging mixture (available from Thermo Scientific).

[0037]The results of these studies are shown in FIG. 6 which shows that over-expression of TSG101, as well as Annexin 2 increase in viral titers.

[0038]The studies described above have identified mu...

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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.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application claims the benefit of the filing date of U.S. Provisional Application Ser. No. 61 / 388,338, filed Sep. 30, 2010, the entire disclosure of which is incorporated by reference as if set forth fully herein.FIELD OF INVENTION[0002]The present invention relates to lentivirusesBACKGROUND OF INVENTION[0003]Lentiviruses (LVs) designed to deliver transgenic cargo (e.g., a protein coding sequence or an shRNA) are valuable tools in basic research, bioproduction, and therapeutic delivery. In order to generate these valuable reagents, researchers generally introduce a transfer vector (encoding the desired transgenic viral genome to be packaged) along with one or more packaging vectors (that produce essential viral proteins) into an appropriate packaging cell line (e.g. HEK 293 cells). The inventors have recognized that the current set of reagents and protocols is highly inefficient and that new methods are needed to develop sufficient qu...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N5/02
CPCC12N15/86C12N2740/15052C12N2740/15051C12N15/113
Inventor SPAYD, KATIE JANSENKARPILOW, JONWAKEFIELD, JOHN K.
Owner DHARMACON INC
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