A hybrid vector system expands adeno-associated viral vector packaging capacity in a transgene-independent manner

The drastic differences in HB reconstitution within AP intron sequence seen between different studies may reflect undetected or unreported mutations between sequences, especially in the ITRs, indicating sensitivity of reconstitution to minor variations.

“While all designs seemingly use sequences originally reported by Ghosh et al. (2008), the drastic differences in HB reconstitution within AP intron sequence seen between the different studies, may reflect undetected or unreported mutations between sequences, especially in the ITRs, indicating sensitivity of reconstitution to minor variations (Ghosh et al., 2008).”

The hybrid dual AAV strategy was initially proposed by Gosh and colleagues in 2008 and found to significantly improve transgene reassembly in a variety of studies thereafter.

“The hybrid dual AAV strategy was initially proposed by Gosh and colleagues in 2008 [54] and found to significantly improve transgene reassembly in a variety of studies thereafter [29,49].”

Even when only 1,000 vector genomes per cell were used, the transduction efficiency of the hybrid vectors reached 71% of the single vector with a genome size fitting into a single AAV capsid.

“Even when only 1,000 vector genomes per cell were used, the transduction efficiency reached 71% of the single vector with a genome size fitting into a single AAV capsid. 65”

The hybrid vector outperformed both the overlapping and trans-splicing variants, which only expressed levels of approximately 25% and 50%, respectively, of the original construct.

“Thus, the hybrid vector outperformed both the overlapping and trans-splicing variants, which only expressed levels of approximately 25% and 50%, respectively, of the original construct.”

When β-galactosidase activity from muscle tissue was quantified, the hybrid vectors yielded 81% of the activity of the single regular size genome vector, which was significantly greater than the β-galactosidase activity from overlapping (34%) or trans-splicing vectors (62%).

“When b -galactosidase activity from muscle tissue was quantified, the hybrid vectors yielded 81% of the activity of the single ''regular size genome'' vector, which was significantly greater than the b -galactosidase activity from overlapping (34%) or trans-splicing vectors (62%).”

When MO59K cells were transduced with the hybrid vectors at a multiplicity of infection of 20,000 viral genomes per cell, LacZ expression from the hybrid vectors was equal to that of the single intact vector.

“When MO59K cells were transduced with the hybrid vectors at a multiplicity of infection of 20,000 viral genomes per cell (10,000 vector genomes of each vector), the LacZ expression from the hybrid vectors was equal to that of the single intact vector.”

The size of the AP sequence (0.87 kb within each vector) was a burden on the packaging capacity of the two vectors, effectively reducing the combined genome capacity of the dual-vector system from 10 to 8.2 kb.

“However, the size of the AP sequence (0.87 kb within each vector) was a burden on the packaging capacity of the two vectors, effectively reducing the combined genome capacity of the dual-vector system from 10 to 8.2 kb. 65”

Expression from the single vector with a regular size genome and the hybrid vectors was strong and widespread, whereas expression from the trans-splicing, and especially the overlapping vectors, was not as effective.

“Expression from the single vector with a ''regular size genome'' and the hybrid vectors was strong and widespread, whereas expression from the transsplicing, and especially the overlapping vectors, was not as effective.”