Methods for determining potency of adeno-associated virus preparations

A preparation and dosage technology, which is applied in the field of determining the potency of adeno-associated virus preparations, and can solve problems such as wrong dosage of patients

Pending Publication Date: 2019-10-18
TAKEDA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

If dose and / or potency cannot be accurately determined, patients may receive the wrong dose and / or widely varying doses from treatment to treatment

Method used

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  • Methods for determining potency of adeno-associated virus preparations
  • Methods for determining potency of adeno-associated virus preparations
  • Methods for determining potency of adeno-associated virus preparations

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0121] This example illustrates an AAV-specific ELISA. ELISA determines the amount of AAV8 particles present in assay fractions or preparations.

[0122] An AAV-specific ELISA was performed to identify AAV-containing fractions. Conformation-specific monoclonal antibodies were used to detect epitopes repeatedly expressed on assembled AAV-8 particles.

[0123] AAV8 ELISA was performed on a TECAN Roboter system with the AAV-8 titration ELISA kit (Cat. No. PRAAV8; Progen (Heidelberg, Germany)). Briefly, monoclonal antibodies specific for conformational epitopes on the assembled AAV8 capsid (ADK8) were coated on microtiter strips and used to capture AAV8 particles from AAV fractions. Captured AAV8 particles were detected by two steps. In the first step, a biotin-conjugated monoclonal antibody (specific for ADK8 antibody) binds to the immune complex (ADK8 and ADK8 antibody). Add streptavidin peroxidase conjugate to the immune complex bound to biotin-conjugated monoclonal antibod...

Embodiment 2

[0126] This example demonstrates cryogenic transmission electron microscopy (CryoTEM).

[0127] CryoTEM enables visualization of nanoparticles such as AAV. The technique involves depositing AAV formulations on thin supported carbon films in a temperature and humidity controlled environment. After removing excess AAV formulation (leaving some adhered AAV formulation), grids were vitrified in liquid ethane and then stored in liquid nitrogen. CryoTEM analysis of AAV capsid particles was used to assess overall sample morphology, i.e., the presence of various AAV morphologies (often including spherical and deformed AAV particles, subunit structures, and larger structurally less defined morphologies) by automated image analysis methods. ), and the packing level of the pellets.

[0128] AAV fractions or formulations are kept close to their native state by embedding them in amorphous amorphous ice on an inert carrier by flash freezing under cryogenic conditions. Image-based analysi...

Embodiment 3

[0132] This example demonstrates that using an AAV-specific ELISA results in less variability than using qPCR technology.

[0133] AAV8 ELISA: Total AAV8 capsid particle titers were measured using a commercial kit (Progen) calibrated to capsid particle (cp) concentration as described in Example 1. AAV8 reference standard material.

[0134] qPCR: developed based on Chemical quantitative PCR assay to measure vector genome copy concentration of transgenic DNA. For ITR-qPCR of FVIII, the vector genome titer (vg) per milliliter (ml) was determined using TaqMan-based qPCR (using primers and fluorescently labeled probes to detect sequences within the ITR sequence of the vector genome). To detect ITR-specific sequences in AAV particles, samples were treated with DNAse I, and after a subsequent proteinase K step, the scAAV genome was released from the capsid. Finally, restriction enzyme digestion was performed to resolve the AAV ITR T-type structure.

[0135] A plasmid used as a ...

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Abstract

Provided herein are methods of measuring the qualitative and / or quantity attributes of gene therapy vector preparations. In certain embodiments, the gene therapy vector preparations are Adeno-associated virus (AAV) preparations (e.g., AAV8). In certain embodiments the methods comprise determining the potency or dose of the AAV preparation using ELISA, or ELISA in combination with cryogenic transmission electron microscopy (CryoTEM).

Description

[0001] related application [0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 467,045, filed March 3, 2017, the contents of which are incorporated herein by reference in their entirety. Background technique [0003] Adeno-associated virus (AAV, adeno-associated virus) is a small non-enveloped virus that packages a linear single-stranded DNA genome. AAV belongs to the family Parvoviridae and the genus Dependovirus, since productive infection with AAV occurs only in the presence of a helper virus such as adenovirus or herpes virus. Even in the absence of helper virus, AAV (serotype 2) can achieve latency by integrating into chromosome 19q13.4 of the host human genome. It is the only mammalian DNA virus known to be capable of site-specific integration (Daya and Berns, Clinical Microbiology Reviews, pp. 583-593 (2008)). [0004] In order to use AAV safely in the clinic, AAV has been genetically modified at several locations within its genome....

Claims

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Application Information

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IPC IPC(8): G01N33/569C12N15/864G01N33/48
CPCG01N33/48G01N33/56983G01N2333/015C12N15/86C12N2750/14143C12N2750/14151G01N33/4833C12N15/8645A61K48/0091C12N7/00G01N1/36G01N1/42
Inventor M·本迪克R·尼西纳E·伯姆M·格兰因戈尔C·菲德勒
Owner TAKEDA PHARMA CO LTD
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