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Nucleic acid construct

A nucleic acid construct and nucleic acid sequence technology, applied in the field of relative expression constructs, can solve the problems of difficult prediction and adjustment of expression ratio and inability to control relative expression, etc.

Inactive Publication Date: 2018-01-02
AUTOLUS LIMIED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A key limitation of this approach is the inability to control the relative expression
3' transcripts are generally expressed less than 5' transcripts, but the expression ratio is difficult to predict and adjust
The problem with using 2A peptides for cleavage between different peptides in the same ORF is that expression is limited to a 1:1 ratio

Method used

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Examples

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Effect test

Embodiment 1

[0212] Example 1 - Using the mouse Ig kappa chain V-III signal sequence

[0213] PCT / GB2014 / 053452 describes a vector system encoding two chimeric antigen receptors (CARs), one for CD19 and one for CD33. The signal peptide used for CAR in this study was the signal peptide from the human CD8a signal sequence. For the purposes of this study, this was replaced with a signal peptide from the V-III region of the murine Ig kappa chain with the following sequence: METDTLILWVLLLLVPGSTG (hydrophobic residues are highlighted in bold). In order to establish that the murine Ig kappa chain V-III signal sequence functions as well as the signal sequence from human CD8a, comparative studies were performed. Functional expression of anti-CD33 CAR and anti-CD19 CAR was observed for both signal sequences. This substituted signal sequence and all subsequent mutations were transiently transfected into 293T cells. Three days after transfection, 293T cells were stained with both soluble chimeric...

Embodiment 2

[0214] Example 2 - Alteration of Relative Expression by Deletion of Hydrophobic Residues in Signal Peptides

[0215] Hydrophobic residues were deleted in a stepwise manner and the effect on the relative expression of anti-CD33 CAR and anti-CD19 CAR was observed. The effects of one, two, three and four amino acid deletions were studied and the results were as follows Figure 5 to Figure 8 shown.

[0216] All mutant constructs showed reduced relative expression of the anti-CD19 CAR compared to the anti-CD33 CAR. The greater the number of amino acid deletions from 1 to 3, the greater the relative reduction in anti-CD19 CAR expression, but then plateaued (four deletions produced a similar reduction in expression as three deletions).

[0217] Thus, modification of the signal sequence in the nucleic acid constructs encoding the two polypeptides can be used to control the relative expression of the two polypeptides.

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Abstract

The present invention provides a nucleic acid construct comprising the following structure: A-X-B in which A is nucleic acid sequence encoding a first polypeptide which comprises a first signal peptide; B is nucleic acid sequence encoding a second polypeptide which comprises a second signal peptide and X is a nucleic acid sequence which encodes a cleavage site, wherein the first signal peptide orthe second signal peptide comprises one or more mutation(s) such that it has fewer hydrophobic amino acids.

Description

field of invention [0001] The present invention relates to constructs and methods for modulating the relative expression of polypeptides co-expressed from a single vector. In particular, the invention relates to modulating the expression of a transmembrane protein co-expressed with a second polypeptide from a single vector. Background of the invention [0002] It is often desirable to express different proteins from the same vector, as multiple transduction of the same cell is difficult, expensive and unpredictable. Different methods have therefore been developed to allow co-expression of the two proteins from a single vector (see figure 1 ). [0003] Initial attempts used two different promoters in the same cassette. This produces two separate transcripts, each encoding a separate protein. This is the difficult method for several reasons. A key issue is "promoter interference," in which one promoter takes over and causes silencing of a second promoter. Furthermore, di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N5/10
CPCC07K16/2803C07K2319/02C07K14/7051C07K14/70517C07K14/70521C07K14/70589C12N5/0636C12N2510/00C07K2319/33C07K14/685C12N9/16C12Y301/03048C07K2319/03A61K39/0011A61K2039/5156A61K2039/5158C07K7/08C07K2317/622C07K2319/30C07K2319/50C07K2319/70
Inventor M.普莱S.科多巴
Owner AUTOLUS LIMIED
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