676. DNA Sequences Encoding shRNAs Can Replace Mutant ITR in scAAV Genome for Efficient Replication and Packaging and Transcribe shRNAs by pol III Promoter Activity of wt ITR for Efficient Gene Silencing
Authors: Jun Xie, Qin Mao, Ran He, Ye Zhu, Jia Li, Qin Su, Mengxin Li, Li Zhong, Chris Muller, Heather Zhou, Guangping Gao
Journal: Molecular Therapy (2015)

The two inverted terminal repeats (ITRs) at each end of recombinant adeno-associated virus (rAAV) genome are the only sequences inherited from wild type (Wt) viral genome. They contribute to rAAV genome replication, packaging, intracellular processing and stabilization, and integration into host genome. Previously we showed DNA sequences encoding shRNAs (shDNA) are the barriers in self-complementary AAV (scAAV) genome replication, resulting in vector genome truncations and heterogeneity. We now observe the same phenomena in the genome replication and packaging of single-stranded AAV vectors harboring shDNA. When detected on a denaturing gel, these truncated genomes were doubled in size as seen for scAAV, indicating they are intra-molecular double-stranded DNA molecules. We hypothesized DNA with hairpin structures could serve as the mutant ITR during rAAV genome replication and packaging. To test this, we replaced the mutant ITR in scAAV genome with the DNA encoding shRNA against Apob or Firefly luciferase gene. Southern blot of Hirt's DNAs revealed that vector genomes flanked with shDNA and wtITR, but not with shDNA and mutant ITR, were efficiently rescued and replicated when co-transfected with adeno-helper plasmid and Rep/Cap trans-plasmid into HEK293 cells. Large scale AAV production and analyses of CsCl gradient purified rAAV genomes in both native and denaturing gels further demonstrated that hybrid shDNA-wtITR vector genomes were efficiently packaged into AAV capsid as double-stranded genomes. To test their functionalities in vivo, we intravenously injected 2×1011 GCs of scAAV9 containing shApoB-wtITR genome with an EGFP gene into adult C57 mice and harvested liver tissues 3 weeks later. Compared to regular scAAVEGFP vector, the shApoB-wtITR AAV vectors achieved comparable EGFP transduction efficacy in the liver. Interestingly, without a H1 promoter the shApob produced abundant small antisense RNA and silenced mouse ApoB gene as efficienct as conventional scAAVshRNA vectors. Southern blots of mouse liver detected linear and circularized forms of AAV genomes, thus implying that wtITR in the circularized genome may serve as a strong pol III promoter to drive shRNA expression. Currently, we are in the process of replacing Wt ITR with an artificial chimeric ITR with only rep binding element, terminal resolution sites and D sequence of authentic ITR retained. In summary, hairpin structure sequences can function as an alternative mutant ITR for production of functional scAAV. Our findings may have significant impact on AAV biology and vectorology, potentially enabling us to create artificially intelligent and programmable vector genomes with improved packaging efficacy, expanded genome sizes, modulated tissue tropism and propensity of integration, regulatable in vivo persistence and potency, etc.