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Mammalian cells for producing adeno-associated viruses

A mammalian and adenovirus technology, applied in the direction of viruses, viral peptides, microorganisms, etc.

Pending Publication Date: 2020-03-24
LONZA LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, CHO cells have not been used to produce rAAV before

Method used

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  • Mammalian cells for producing adeno-associated viruses
  • Mammalian cells for producing adeno-associated viruses
  • Mammalian cells for producing adeno-associated viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0176] Example 1: CHOK1SV GS-KO co-infected with adenovirus 5 virus TM Transient AAV production in

[0177] To determine the CHOK1SV GS-KO TM (Lonza, Slough, UK) Can the cells biologically produce and assemble the rAAV carrier protein, the CHOK1SV GS-KO TM Cells were transfected with two plasmids: pRC2-mi342 (6234, Clontech) and pAAV-GFP (AAV-400, Cell Biolabs) control vector (Catalogue Number: AAV-400 Cell Biolabs, San Diego, USA)( Image 6 ), in wt Ad5 virus (catalog number: VR-1516 TM , American Type Culture Collection, Manassas, USA). Under natural conditions, CHOK1SV GS-KO TM Cells do not accept adenovirus due to lack of viral receptors, but in the presence of cationic transfection reagents (Lipofectamine or PEI), negatively charged adenovirus 5 is able to enter CHOK1SV GS-KO TM cells, thereby providing all auxiliary elements. This concept was used to design the evaluation experiment, where CHOK1SVGS-KO TM Cells provided all elements (Rep-Cap and GOI by transfec...

Embodiment 2

[0178] Example 2: In the absence of viral co-infection, CHOK1SV GS-KO TM Transient rAAV production in

[0179] To determine that in the absence of wt Ad5 virus, CHOK1SV GS-KO TM Whether cells can produce rAAV, cells were engineered to express Ad5E1A and E1B using a method similar to Howe et al. (2005). A plasmid construct expressing E1A under the control of the TET-on promoter (pMF30, figure 2 ), expressing E1B under the control of a constitutive human CMV promoter (pMF23, image 3 ), and the Ad5 E1A-E1B open reading frame (pXC17.4_17AV5PerProKZ, Figure 4 ). CHOK1SV GS-KO was established by transfecting with pMF30 and selecting for cells able to survive in glutamine-free medium supplemented with 50 μM L-MSX TM Derived library ( Figure 5 A). CHOK1SV GS-KO was then transfected with pMF23 TM E1A, and select cells that can survive in glutamine-free medium supplemented with 50 μM L-MSX and 10 μg / mL puromycin ( Figure 5 A). The water control was contaminated in all gen...

Embodiment 3

[0180] Example 3: In the absence of viral co-infection, CHOK1SV GS-KO TM and permanent rAAV generation in HEK293.

[0181] Integrate landing pad suitable for subsequent RMCE to CHOK1SV GS-KO TM and HEK293 genomes, as from recombinant CHOK1SV TM Loci identified in cell lines (Fer1L4, NL1, 2, 3, 4, 5 and 6 (see eg WO2013190032A1 and EP2711428A1)). A schematic diagram of the arrangement of components within these landing pads is given in Figure 7 A, B and C are shown. The location of the Frt site between the SV40E promoter and the selectable marker allows it to be used in subsequent RMCE. Use of incompatible Frt sites (A, B, C, D, E, and F) and different reporter / selection markers ensures that the sites are independently addressable. Figure 8 displayed in 1 ( Figure 8 A), 2 ( Figure 8 B) and 3 ( Figure 8 C) Various permutations of AAV, Ad5 and GOI genes after RMCE in locus CHO- and HEK293-derived SSI cell lines.

[0182]To generate the HEK293 SSI host, a BLAT search...

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Abstract

A mammalian cell comprising at least four distinct recombination target sites (RTS), an adenovirus (Ad) gene comprising E1A, E1B or a combination thereof, and a promoter operatively linked to the Ad gene, wherein the RTS, the Ad gene, and the promoter are chromosomally-integrated; methods for using the cell for generating a recombinant adeno-associated virus (rAAV) producer host cell; and methodsfor using the AAV producer host cell to produce, package and purify rAAV.

Description

[0001] field of invention [0002] The present disclosure relates to a mammalian cell comprising at least four different recombination target sites (RTS), an adenovirus (Ad) gene comprising E1A, E1B or a combination thereof, and a promoter operably linked to the Ad gene , wherein the RTS, Ad gene and promoter are chromosomally integrated. The present disclosure also relates to methods of using the cells to generate recombinant adeno-associated virus (rAAV) producer cells, and methods of using the rAAV producer cells to produce, package and purify rAAV in the absence of live helper virus. Background technique [0003] Recombinant adeno-associated virus (rAAV) is being actively used in clinical development for efficient long-term delivery of gene therapy products. Baculovirus-based platforms for rAAV production typically require raw materials that are variable and time-consuming to generate. Poor scalability may limit triple transfection and transient expression in HEK293. Ex...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N7/00C12N15/86C12N15/90
CPCC12N15/86C12N15/90C12N2750/14152C12N2800/30C12N2999/007C07K14/005C12N2710/10322C12N7/00C12N2750/14143
Inventor M·福瑞R·J·杨A·塞思
Owner LONZA LTD
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