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Methods of heat inactivation of adenovirus

A technology of adenovirus and virus, which is applied in the field of thermal inactivation of adenovirus, can solve the problems of AAV vector genome damage or degradation, reducing the quality and quantity of AAV vector, etc.

Pending Publication Date: 2019-01-11
ULTRAGENYX PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, commonly used helper virus inactivation methods, such as heat inactivation, can also lead to disruption or degradation of the AAV vector genome, thereby reducing the quality and quantity of AAV vector that can be recovered from the inactivation process

Method used

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  • Methods of heat inactivation of adenovirus
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  • Methods of heat inactivation of adenovirus

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Experimental program
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preparation example Construction

[0037] Preparation of AAV Vectors

[0038] AAV vectors can be prepared in mammalian or insect cells by a variety of methods known in the art. Any production method will be suitable for producing the starting material of the present invention as long as the result of the production method is a sample containing AAV and helper virus. The manufacturing process involves providing cells with a plasmid containing the AAV vector genome, with AAV rep and cap gene functions, and additional helper functions. Other helper functions can be provided, for example, by AV infection, by plasmids carrying all necessary AV helper function genes, or by other viruses such as HSV or baculovirus. AAV production methods suitable for use in the methods of the present invention include Clark et al., Human Gene Therapy 6: 1329-1341 (1995); Martin et al., Human Gene Therapy 24: 253-269 (2013); Thorne et al., Human Gene Therapy 20: 707 -714 (2009); Fraser Wright, Human Gene Therapy 20:698-706 (2009); Vi...

Embodiment 1

[0062]Ad5AV particles were produced using standard techniques and minimally purified by CsCl gradients. Ad5 particles were dialyzed into background buffer (40 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, 0.001% w / v) Pluronic F68 , pH 8.0). Heat using a thermal cycler to immediately heat the sample to the desired temperature, hold it at the desired temperature, and then immediately cool the sample. Ad5 particles in buffer were contained in polypropylene tubes in volumes below 300 μL. Ad5AV samples were heated to 45°C, 47°C, 49°C or 51°C for 10 minutes, 15 minutes or 40 minutes, except for Ad5AV samples heated to 51°C, which were held at that temperature for 10 minutes only. After heat treatment, use QuickTiter according to manufacturer's instructions TM Ad5 titration immunoassay kit (Cell Biology Laboratories, San Diego, CA) measures Ad5 infectivity.

[0063] figure 1 Heat inactivation data for Ad5AV is shown, shown as Log Rem...

Embodiment 2

[0065] Detect different serotypes and AAV vectors containing different genome types and sizes, and test the effect of AV heat inactivation protocols on the stability of AAV genomes. Two serotypes, AAV8 and hu37, and three different genomes were tested. The following are the detection of AAV vectors:

[0066] (A) hu37 capsid containing single-chain construct (4.7kb)

[0067] (B) hu37 capsid (5.1 kb) containing the oversized single-chain construct

[0068] (C) AAV8 capsid containing a small single-chain construct (4.1kb)

[0069] (D) AAV8 capsid containing self-complementary construct (4.6kb)

[0070] AAV vectors (A) (C) and (D) were prepared in HEK293 cells and particles (B) were prepared in HeLa cells. All supports were minimally purified by affinity chromatography or iodine gradients using standard techniques. Vehicle was dialyzed into background buffer (40 mM 1,3-bis[tris(hydroxymethyl)methylamino]propane, 20 mM HEPES, 20 mM citrate, 200 mM NaCl, 0.001% (w / v) Pluronic F...

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Abstract

The present disclosure generally relates to methods of protecting the genomic integrity and / or biological activity of AAV viral particles in a sample containing both AAV particles and helper virus particles during heat inactivation. The methods include heating, to a temperature greater than or equal to 45 DEG C, a sample containing helper virus particles, AAV particles, and a buffer. The buffer includes a concentration of 10 mM or greater kosmotropic salts and / or a concentration of 10 mM or greater of divalent or trivalent cations.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the benefit of priority to US Provisional Application Serial No. 62 / 314,116, filed March 28, 2016, entitled "Method of Heat Inactivation of Adenovirus," the entire contents of which are incorporated herein by reference in their entirety. Background technique [0003] Adeno-associated virus (AAV) is a non-pathogenic, replication-defective parvovirus. AAV vectors have many unique features that make them attractive as vectors for gene therapy. Specifically, AAV vectors are capable of delivering therapeutic genes to dividing and non-dividing cells, and these genes can persist for extended periods of time without integrating into the target cell's genome. However, in order to make AAV vectors, helper virus functions must be provided, sometimes in the form of active infectious viruses such as adenovirus (AV). To isolate therapeutic AAV vectors, helper virus particles must be inactivated during AAV vect...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N15/864C12N7/04
CPCC12N7/00C12N15/86C12N2710/10361C12N2750/14143C12N2750/14151A61K35/761C12N15/85C12N2523/00
Inventor 克里斯托弗·J·默里森詹姆士·D·马雷特
Owner ULTRAGENYX PHARMA
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