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Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors
Published on: January 29, 2019
Characterization of genome integrity for oversized recombinant AAV vector
Biao Dong1, Hiroyuki Nakai, Weidong Xiao
1The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Molecular Therapy : the Journal of the American Society of Gene Therapy
|November 12, 2009
Summary
Recombinant adeno-associated virus (rAAV) gene therapy can overcome packaging limits. Oversized rAAV vectors, though produced in lower yields, enable transgene expression by complementary genome fragments.
Area of Science:
- Molecular Biology
- Gene Therapy
- Virology
Background:
- Recombinant adeno-associated virus (rAAV) vector packaging capacity is a key limitation in gene therapy.
- Previous research indicates rAAV can achieve persistent transgene expression exceeding its typical 4.7-kb packaging limit.
Purpose of the Study:
- To investigate the mechanism by which oversized rAAV vectors achieve transgene expression.
- To analyze the integrity and recovery of rAAV genomes post-transduction.
Main Methods:
- Construction of rAAV vectors with genomes ranging from 2.9 to 7.2 kb.
- Production and yield assessment of rAAV vectors of varying sizes.
- Analysis of packaged genome integrity using alkaline Southern blot hybridization.
- Rescue and characterization of circularized rAAV genomes from transduced cells and in vivo mouse models.
Main Results:
- rAAV production yields decreased significantly for oversized vectors (>4.7 kb).
- Packaged genomes from oversized vectors were often truncated, but circularized monomers could be rescued post-transduction.
- Larger circularized rAAV genomes were recovered in vivo from oversized vectors, suggesting genome restoration.
Conclusions:
- Partially packaged rAAV sequences can potentially complement each other to restore a full expression cassette.
- This mechanism may enable gene therapy applications with larger therapeutic transgenes using rAAV vectors.
