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Size and Structure of Viral Genomes01:26

Size and Structure of Viral Genomes

Viral genomes exhibit remarkable diversity in size, structure, and composition, influencing their replication strategies and interactions with host cells. These genomes consist of either DNA or RNA and may be linear or circular. Additionally, they can be single-stranded or double-stranded, with each configuration affecting how the virus propagates within a host. RNA viruses, for instance, generally have smaller genomes than DNA viruses, a factor that contributes to their high mutation rates and...

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Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models
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Effect of genome size on AAV vector packaging.

Zhijian Wu1, Hongyan Yang, Peter Colosi

  • 1Ocular Gene Therapy Laboratory, Neurobiology-Neurodegeneration and Repair Laboratory, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

Molecular Therapy : the Journal of the American Society of Gene Therapy
|November 12, 2009
PubMed
Summary

Adeno-associated virus (AAV) vector genomes are limited to 5.2 kb, regardless of plasmid size. Larger vectors yield truncated genomes that can express genes in vitro at high multiplicities of infection (MOI) via recombination.

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Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus (AAV) Capsid Variants

Published on: October 18, 2022

Area of Science:

  • Molecular Biology
  • Virology
  • Gene Therapy

Background:

  • Adeno-associated virus (AAV) vectors are crucial for gene therapy, but their packaging capacity is traditionally limited.
  • Previous studies suggested AAV vector genomes could not exceed the size of the parental AAV genome, typically around 5 kilobases (kb).
  • Recent claims proposed the possibility of packaging significantly larger AAV vector genomes intact.

Purpose of the Study:

  • To investigate the actual packaging limits of adeno-associated virus (AAV) vector genomes.
  • To determine if larger plasmid-encoded AAV vectors can be packaged intact by different AAV serotypes.
  • To analyze the characteristics and functionality of packaged genomes derived from oversized vectors.

Main Methods:

  • Analysis of packaged vector genomes from plasmid-encoded AAV vectors ranging from 4.7 to 8.7 kb.
  • Utilized AAV serotypes 2, 5, and 8 capsids for packaging experiments.
  • Employed Southern blot analysis and strand-specific oligonucleotide probing to characterize packaged genomes.

Main Results:

  • Packaged AAV vector genomes consistently measured no more than 5.2 kb, irrespective of the initial plasmid vector size or AAV capsid type.
  • Vector genomes derived from plasmids exceeding 5 kb were found to be heterogeneous and truncated at the 5' end.
  • Despite truncation, these larger vector preparations mediated reporter gene expression in vitro at high multiplicity of infection (MOI), albeit less efficiently than smaller vectors.

Conclusions:

  • The packaging limit for adeno-associated virus (AAV) vectors is approximately 5.2 kb, challenging previous assumptions about larger genome packaging.
  • Fragmentary genomes from oversized AAV vectors can generate functional, intact genomes within cells at high MOI, likely through recombination.
  • This finding has implications for designing AAV vectors and understanding gene expression from oversized constructs in gene therapy applications.