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Universal self-regulating mammalian cell line platform for the production of biologics

A cell-level technology, applied in the direction of medical preparations containing active ingredients, microorganisms, biochemical equipment and methods, etc., can solve problems such as adverse product results, cell stress, inability to coordinate intracellular state of product gene transcription, etc.

Pending Publication Date: 2020-02-11
LONZA LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such systems fail to coordinate product gene transcription with the intracellular state of the host cell line, as occurs, for example, with endogenous host cell proteins, leading to cellular stress and poor product outcomes, especially for NGB

Method used

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  • Universal self-regulating mammalian cell line platform for the production of biologics
  • Universal self-regulating mammalian cell line platform for the production of biologics
  • Universal self-regulating mammalian cell line platform for the production of biologics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0342] 1. A cell comprising:

[0343] a first control element operably linked to a sequence encoding an exogenous therapeutic polypeptide;

[0344] a second control element operably linked to a sequence encoding a repressor polypeptide; and

[0345] Optionally, a third control element operatively linked to a sequence encoding one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) gRNAs;

[0346] in:

[0347] i) the second control element has a first level of activity under the first condition and a second level of activity under the second condition; or

[0348] ii) the third control element has a first level of activity under the first condition and a second level of activity under the second condition, and

[0349] Wherein, in the presence of the second condition, expression of the therapeutic polypeptide is modulated.

[0350] 2. A cell comprising:

[0351] a first control element operatively linked to the insertion site;

[0352] a second control element operably l...

Embodiment 1

[0584] In the example below, using Figure 1A and 1B Design principles for the genetic control loop shown in . In this example, using Figure 3A The circuit test shown utilizes the principle that a repressor (i.e., dCas9) inhibits the expression of a recombinant protein gene (i.e., GFP). The existing CHOK1SV derived GS-KO (Xceed TM ) cell line, the recombinant polypeptide gene GFP is operatively linked to a first control element (for example, a first promoter element, hCMV), which is transiently transfected with: 1) expression of only encoding repressor polypeptide dCas9 Vector, the dCas9 is operatively linked to a constitutive mCMV promoter, or 2) an expression vector encoding a repressor polypeptide (dCas9) plus a vector expressing gRNAs 1, 2 and 3, each controlled by a separate U6 promoter ( Figure 3A ). Four days after transfection, GFP fluorescence was determined by flow cytometry and it was observed that transfection with dCas9+gRNA 1-3 resulted in population GFP co...

Embodiment 2

[0586] In this example, using Figure 4A The circuit shown tests the principle of using a repressor (i.e., dCas9) to suppress gene expression of recombinant mAb heavy chain (HC) and light chain (LC). In this example we demonstrate that the therapeutic polypeptide IgG4 Mab operably linked to a first control element (e.g. the promoter element hCMV) is inhibited by the inhibitory polypeptide dCas9 operably linked to the constitutive mCMV promoter Expression of cB72.3. Using a library of CHO cell lines stably expressing IgG4 Mab cB72.3 HC and LC genes each driven by a separate hCMV promoter, the ability to downregulate Mab expression using dCas9 and gRNAs 1-3 targeting the hCMV promoter was tested. The library uses only dCas9 plasmid (dCas9) or dCas9 plasmid + / - gRNA encoding plasmid (dCas9+g1-g3, see Figure 4B ) was transiently transfected, and the Mab concentration was determined using an Octet bioanalyzer on days 3, 4, and 5 after transfection ( Figure 4B ). Error bars re...

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Abstract

Disclosed are genetic control circuits, cells, and methods that use a repressor polypeptide to reduce the transcription rate of an exogenous therapeutic polypeptide encoding gene in response to a change in condition.

Description

[0001] related application [0002] This application claims priority to U.S. Serial No.: 62 / 521,005, filed June 16, 2017, the entire contents of which are incorporated herein by reference. [0003] field of invention [0004] The present invention relates to methods and compositions for modulating the expression of products, such as recombinant proteins, by cells and cell lines utilizing genetic control circuits responsive to cellular stress. Background technique [0005] Recombinant therapeutic proteins are typically expressed in cellular expression systems, such as mammalian cell expression systems. In 2014, the total number of biologic drugs approved for the market was 212, and 56% of the therapeutic products approved by the FDA to be placed on the market were produced in mammalian cell lines. However, high costs associated with production lead to increased global health costs. [0006] In addition, next-generation protein biologics (NGB), such as next-generation fusion ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113
CPCC12N15/113C12N9/22C12N15/11C12N15/85C12N2800/80A61K39/00
Inventor R·杨P·M·奥卡拉汉T·佩恩
Owner LONZA LTD
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