Effect of genome size on AAV vector packaging.

By Zhijian Wu, Hongyan Yang, P. Colosi. Molecular Therapy, 2010

917 citations recorded by the source corpus.

Adeno-associated virus (AAV) vector genomes have been limited to 5 kilobases (kb) in length because their packaging limit was thought to be similar to the size of the parent AAV genome. Recent reports claim that significantly larger vector genomes can be packaged intact. We examined the packaged vector genomes from plasmid-encoded AAV vectors that ranged from 4.7 to 8.7 kb in length, using AAV types 2, 5, and 8 capsids. Southern blot analysis indicated that packaged AAV vector genomes never exceeded 5.2 kb in length irrespective of the size of the plasmid-encoded vector or the capsid type. This result was confirmed by vector genome probing with strand-specific oligonucleotides. The packaged vector genomes derived from plasmid-encoded vectors exceeding 5 kb were heterogeneous in length and truncated on the 5' end. Despite their truncated genomes, vector preparations produced from plasmid-encoded vectors exceeding 5.2 kb mediated reporter gene expression in vitro at high multiplicity of infection (MOI). The efficiency of expression was substantially lower than that of reporter vectors with genomes <5 kb in length. We propose that transcriptionally functional, intact vector genomes are generated in cells transduced at high MOI from the fragmentary genomes of these larger vectors, probably by recombination.

Subfields: Epidemiology, Genetics, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/7fd983977204ce0ab2a703fea24d61bd7132f55a

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Citations in the corpus

CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling (2014, background): “Commonly used delivery systems based on lentiviral and ad- eno-associated viral (AAV) vectors have limited packaging ca- pacity (Kumar et al., 2001; Wu et al., 2010), which renders it challenging for incorporation of Cas9 along with sgRNA expres- sion cassettes and necessary genetic elements (i.e.,…”
Optical Control of Mammalian Endogenous Transcription and Epigenetic States (2013, methodology): “In order to use AAV as a vector for the delivery of LITE components, we needed to ensure that the total viral genome size of each recombinant AAV, with the LITE transgenes included, did not exceed the packaging limit of 4.8 kb 31 .”
CRISPR technology: A decade of genome editing is only the beginning (2023, background): “However, one main limitation of AAVs is their low packaging capacity (173).”
Delivering CRISPR: a review of the challenges and approaches (2018, background): “While this is within the realm of technical possibility, the SpCas9 and sgRNA are roughly 4.2kB in size, and the overall size of AAV ( 20 nm) only allows for 4.5–5 kb of genomic material to be packaged within it (Wu et al., 2010).”