763. Size-Dependent Packaging and Transgene Expression of Double-Stranded AAV Vectors

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Efforts to improve the potency of AAV mediated gene transfer should greatly benefit its application as a gene transfer vector, especially in human gene therapy applications, in which large amounts of viral particles are usually required to achieve the desirable therapeutic effects. A genome-modified strategy recently demonstrated by us and others dramatically accelerated and improved AAV mediated gene transfer, holding great promise. This strategy is characterized by rendering AAV vectors predominantly packaging a double-stranded (ds) DNA as genome via mutating one inverted terminal repeat (ITR). In current studies, we further attempted to define the optimal packaging capacity of dsAAV. Vectors of various length between the two ITRs arranging from 1871 base pairs (bp) to 6362 bp were characterized by Southern analysis. We found that the packaging of dsAAV vector is size dependent, with dsAAV vectors less than its packaging capacity (around 2440 bp-2487 bp) predominantly encapsulated full- length ds DNA as their genomes. However, the genomes of over-sized dsAAV vectors were also packaged, containing a smear of partially packaged DNA molecules. The DNA sizes spanned from corresponding single-stranded length to around 4.8 kilonucleotides, dominated by single-stranded DNA molecules and certain length of partial ds DNA molecules. The results suggest that AAV packaging machinery attempts to use sub-optimal excision sites to remove the excessive DNA in the absence of a wild type ITR and trs, prior to the completion of viral particle assembly. In vitro and in vivo infection studies showed that the over-sized, partial dsAAV vectors still exhibited higher transgene expressions in vitro and in vivo than the control single-stranded AAV vector, albeit lower than the fully dsAAV vectors. These findings should have important implications for elucidating the packaging process of AAV virions as well as on the usage of dsAAV as a gene delivery vector.