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Ultra-high-precision viral vector assay

A virus and vector technology, applied in the direction of virus/phage, virus, double-stranded DNA virus, etc., can solve the problem of insufficient sensitivity of roller bottle assay.

Pending Publication Date: 2021-12-31
TRIZELL LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When comparing alternative assays to the industry standard roller bottle assay, we were surprised to find that, contrary to what is taught in the art, the roller bottle assay is not sensitive enough to measure 3 x 10 10 <1 RCA in virions
In contrast, we found that the roller bottle assay can only measure 3 × 10 10 ≥75 RCAs in virions

Method used

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  • Ultra-high-precision viral vector assay
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Embodiment Construction

[0022] Our assay is outlined in figure 1 middle. exist figure 1 , suspension cultured HeLa cells were used to expand possible RCAs. Cells were divided into 7.5×10 7 Cells / flask were inoculated into 500ml shake flasks. Add 3 × 10 of the test sample (TS) to each shake flask 10 virus particles (Vp). Incubate (+37°C, 5% CO 2 , 122 rpm) after three days, the cells were harvested and lysed with three freeze-thaw cycles. The lysate was clarified by centrifugation.

[0023] The lysate was then added to cells with fresh assay cells (2 × 10 7 cells / flask) in a 125ml shake flask. These first assay flasks were treated in the same manner as the target cell cultures.

[0024] The lysate from the first assay culture was then added to the second assay culture (2 × 10 7 cells / flask). After three days of incubation, the second assay culture cells were harvested and lysed. Lysates were clarified by centrifugation and stored in an ultra-low temperature freezer. Multiple cycles of RC...

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Abstract

During manufacture of replication-deficient viral gene therapy vector, random mutation or other events may produce undesirable replication-competent virus ("RCV"). Viral gene therapy vector manufacturers thus assay for the presence of contaminating RCV by assaying for serial infection, i.e., transducing target cells with the viral vector, and then lysing the transduced cells, and then mixing the lysate with live assay cells, and then microscopically observing the assay cells to visually determine whether they have been infected with virus. We have tested various alternative approaches, and surprisingly found that droplet digital PCR is not only faster than the prior art approach, but is also over an order of magnitude more sensitive, able to detect, for example, in 3 x 1010 assay cells, as few as seven (7) replication competent adenoviruses ("RC").

Description

[0001] Cross references to related applications: [0002] This application claims priority from US Invention Patent Application Serial No. 16 / 426,124, filed May 30, 2019 and incorporated herein by reference. [0003] Statement Regarding Federal Sponsorship of Research or Development: [0004] Not applicable [0005] Names of parties to the joint research agreement: [0006] Not applicable [0007] Sequence listing reference: [0008] This application includes and is incorporated by reference into the Patent Incorporated in this application TM document. [0009] Statements Regarding Prior Publication of Inventors or Co-Inventors: [0010] Not applicable Background technique: [0011] Certain viral gene therapy vectors are designed not to replicate in patients. For example, to replicate in normal human cells, adenoviruses require functional E1a, E1b, and E3 genomic regions. By deleting or mutating these regions, viral gene therapy vectors can be rendered replication-def...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/86C12Q1/6876C12Q1/686C12Q1/70
CPCC12Q1/70C12Q1/701C12N2710/10343A61K48/0091C12Q1/6888C12Q1/686C12Q2600/106C12Q2600/156C12Q2531/113
Inventor J·迈克南H·佩尔托宁M·哈西宁S·伊拉-赫图拉N·帕克尔
Owner TRIZELL LTD