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Novel adeno-associated virus (AAV) vectors, aav vectors having reduced capsid deamidation and uses therefor

A deamidation and capsid technology, applied in isoaspartic acid, asparagine is deamidated to aspartic acid, mutual transfer, molecular field, can solve problems such as complex drugs and poorly understood strategies

Pending Publication Date: 2021-02-09
THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

AAV capsid post-translational modifications (PTMs) are largely unexplored, and thus, little is known about their potential to affect function or about strategies to control PTM levels in manufactured AAV therapeutics
[0005] Variations in the post-translational modifications of non-gene therapy protein therapeutics complicate their development as drugs

Method used

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  • Novel adeno-associated virus (AAV) vectors, aav vectors having reduced capsid deamidation and uses therefor
  • Novel adeno-associated virus (AAV) vectors, aav vectors having reduced capsid deamidation and uses therefor
  • Novel adeno-associated virus (AAV) vectors, aav vectors having reduced capsid deamidation and uses therefor

Examples

Experimental program
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example

[0202]The following examples report extensive deamidation of AAV8 and 7 additional different AAV serotypes and supporting evidence from structural, biochemical, and mass spectrometry methods. The degree of deamidation at each site depends on the age of the vector and a number of main sequence and 3D structural factors, but it is largely not restricted by the conditions of vector recovery and purification. It is proved that deamidation may affect vector transduction activity, and the loss of the early time point of vector activity is related to the spontaneous deamidation that progresses rapidly at several AAV8 asparagines. A mutation strategy for stabilizing side chain amides was explored to improve vector transduction and reduce batch-to-batch molecular variability, which is a key issue in biological preparation. This study demonstrates previously unknown aspects of AAV capsid heterogeneity and highlights its importance in the development of these vectors for gene therapy.

[0203]The...

example 4

[0204]Example 4 relates to a novel epitope mapped on the AAV9 capsid.

example 1

[0205]Example 1: Deamidation of amino acids on the surface of the adeno-associated virus capsid

[0206]A. Materials and methods

[0207]1.1D and 2D gel electrophoresis

[0208]In order to perform 1D SDS polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the AAV vector was first denatured at 80°C for 20 minutes in the presence of lithium dodecyl sulfate and a reducing agent. Then, it was run on a 4-12% Bis-Tris gel at 200V for 90 minutes and stained with coomassie blue. forFigure 1A-Figure 1DThe data in Kendrick Laboratories, Inc. (Madison, Wisconsin) performed 2D gel electrophoresis. For follow-up experiments, 2D SDS-PAGE was performed internally. To this end, 3×1011GC's AAV vector and 500Uturbonuclease marker (Accelagen, San Diego, CA) in 150μL containing 35mM NaCl and 1mM MgCl2The phosphate buffered saline (PBS) was combined together and incubated at 37°C for ten minutes. Next, add nine volumes of absolute ethanol, vortex the sample, and then incubate it at -80°C for at least two hou...

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Abstract

A recombinant adeno-associated virus (rAAV) vector comprising an AAV capsid having a heterogeneous population of vp1 proteins, a heterogeneous population of vp2 protein and a heterogeneous populationof vp3 proteins. The capsid contains modified amino acids as compared to the encoded VP 1 amino acid sequence, the capsid containing highly deamidated asparagine residues at asparagine-glycine pair, and further comprising multiple other, less deamidated asparagine and optionally glutamine residues. Methods of reducing deamidation in the AAV capsid of a rAAV are provided.

Description

[0001]Statement on Federal Government Funding of Research[0002]The present invention was made with government support under the authorization number P01HL059407 granted by the National Heart, Lung, and Hematology Institute of the National Institutes of Health. The government has certain rights in the invention.Background technique[0003]The structure of the adeno-associated virus (AAV) capsid is an icosahedron and contains 60 viral protein (VP) monomers (VP1, VP2, and VP3) at a ratio of 1:1:10 (Xie Q et al., "American National Bulletin of the Academy of Sciences (ProcNatl Acad SciUSA) 2002; 99(16): 10405-10). Both VP1 and VP2 contain the entire VP3 protein sequence (519aa) in the C-terminus, and the shared VP3 sequence is mainly responsible for the overall capsid structure. Due to the structural flexibility of the unique region of VP1 / VP2 and the low presentation of VP1 and VP2 monomers relative to VP3 monomers in the assembled capsid, VP3 is the only capsid protein that can be disti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/861
CPCC12N15/86C12N2750/14143C12N2750/14122C12N2750/14121C07K14/005C12N7/00
Inventor J·M·威尔逊A·特普K·特纳J·J·辛斯
Owner THE TRUSTEES OF THE UNIV OF PENNSYLVANIA
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