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Adenoviral vectors

An adenovirus and vector technology, applied in the field of adenovirus vectors, can solve problems such as low yield and low transfection efficiency, and achieve economical effects

Pending Publication Date: 2020-03-17
OXFORD GENETICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this approach has many limitations: for example, each batch of AAV and Ad5 particles must be separated to provide a pure product, and it is challenging to ensure that all Ad5 is removed
These include the requirement to pre-prepare sufficient plasmids to transfect into production cell lines, and the inherently inefficient process of transfection itself
The yields of these systems are also lower than those using Ad5-based methods

Method used

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  • Adenoviral vectors
  • Adenoviral vectors
  • Adenoviral vectors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0221] Example 1: Construction of a plasmid vector for expression of a GFP reporter from the Ad5 repressor mutant major late promoter

[0222] Five expression constructs were created, in which the wild-type Ad5 MLP (pMLPwt-GFP), the repressor mutant MLP (pMLP-TET01a-GFP; pMLP-TET01b-GFP; pMLP-TET02-GFP) or the control construct (pMCS- GFP) transcribed the GFP reporter gene, no internal promoter was present in the control construct to determine the low level of baseline transcription. carrier such as Figure 4 shown.

Embodiment 2

[0223] Example 2: Expression of GFP reporter gene from MLP promoter

[0224] Five expression constructs were created, in which the wild-type Ad5 MLP (pMLPwt-GFP), the repressor mutant MLP (pMLP-TET01a-GFP; pMLP-TET01b-GFP; pMLP-TET02-GFP) or the control construct (pMCS- GFP) transcribed the GFP reporter gene, no internal promoter was present in the control construct to determine the low level of baseline transcription. HEK293 cells were seeded at a density of 3e4 cells / well in tissue culture-treated 48-well plates 24 hours before transfection. HEK293 was transfected with plasmids pMLPwt-GFP, pMLP-TET01a-GFP, pMLP-TET01b-GFP, pMLP-TET02-GFP, pMCS-GFP using branched PEI (25kDA) at a ratio of total DNA mass to PEI of 1:3 cell. Transfections were performed in triplicate and cells were harvested for flow cytometry analysis at 24 hours, 48 ​​hours and 72 hours after transfection. data in Figure 5 MFI (Mean Fluorescence Intensity) of GFP-positive cells in . Error bars indicate ...

Embodiment 3

[0225] Example 3: Transcriptional repression of repressor mutant MLP by TETR protein

[0226] Five expression constructs were created, in which the wild-type Ad5 MLP (pMLPwt-GFP), the repressor mutant MLP (pMLP-TET01a-GFP; pMLP-TET01b-GFP; pMLP-TET02-GFP) or the control construct (pMCS- GFP) transcribed the GFP reporter gene, no internal promoter was present in the control construct to determine the low level of baseline transcription. In HEK293 cells treated with doxycycline 0.2 μg / ml or DMSO, wild-type Ad5 MLP expressing GFP reporter (pMLPwt-GFP), repressor mutant MLP at a ratio of 1:1 to total DNA mass (pMLP-TET01a-GFP; pMLP-TET01b-GFP; pMLP-TET02-GFP) or control constructs were co-transfected with a TETR expression plasmid (pTETR) (under the control of a constitutive CMV (cytomegalovirus) promoter). HEK293 cells were seeded at a density of 3e4 cells / well in tissue culture-treated 48-well plates 24 hours before transfection. Transfections were performed in triplicate usin...

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Abstract

The present invention relates to an adenoviral vector comprising a regulatable Major Late Promoter and an exogenous transgene. The invention also provides cells comprising such adenoviral vectors, andprocesses using such vectors.

Description

technical field [0001] The present invention relates to adenoviral vectors comprising a regulatable major late promoter and a foreign transgene. The invention also provides cells comprising such adenoviral vectors and methods of using such vectors. Background technique [0002] Adenoviruses are an attractive and versatile tool in biotechnology, with well-known genetics and the ability to grow to high yields in tissue culture. The replication cycle of adenoviruses is very complex, involving early and late stages. The transition from early to late is thought to occur after DNA replication and activation of the major late promoter (MLP) in the viral genome. MLP drives the expression of all viral late transcripts and can convert up to 30% of cellular proteins into viral structural proteins. In situ modification of MLPs to provide inducible expression has not previously been demonstrated, primarily because the viral DNA polymerase coding sequences are located in opposing DNA s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00
CPCC12N15/86C12N2710/10343C12N2830/005C12N2799/022C12N2810/6018
Inventor 赖安·卡伍德伟恒·苏
Owner OXFORD GENETICS