Surprisingly, these linear DNA molecules integrated in mouse hepatocytes in vivo at a level of 0.3 0.5 vector genome, or more, per diploid genomic equivalent irrespective of the AAV-ITR sequences.
As the parent virus, they carry out vector genome insertion into the host cell chromatin.
These results were further corroborated by quantitation of vector genome copies and histological analysis of reporter (tdTomato) gene expression.
One month post injection, the TA muscle and lumbar spinal cord were analyzed for vector genome copy number and GAA activity.
As expected, with weakly immunogenic hASS, vector genome and hASS expression were sustained with a HDV in spite of ubiquitous expression of the transgene.
We designed the Ad vector genome by insertion of a transgene expression cassette into the E3 region, such that homologous recombination between the Ad vector DNA and 293 cell-derived Ad E1 DNA would produce an Ad vector genome that exceeds in the size of the packaging limit.
Balancing that risk is that the vast majority of integration events seem to be neutral and that optimizing vector design may diminish the probability of altering gene expression by an integrated vector genome.
However, the drop in gene expression observed with the HDV was primarily due to the adaptive immune response, since both β-galactosidase expression and vector genome were sustained in immunodeficient mice treated with HDV.
The total amount of adenoviral vector DNA and the activity of the transferred reporter gene were quantitatively analysed by polymerase chain reaction (PCR) specific for the E4 region of the Ad vector genome and the β-galactosidase assay, respectively.
We constructed ds linear DNA vectors with or without adeno-associated virus inverted terminal repeats (AAV-ITRs), introduced them into mouse hepatocytes in vivo using a hydrodynamics-based transfection technique, and analyzed for vector genome integration in various ways.
However, this construct in its original state was prone to rearrangements of the vector genome, resulting in greatly reduced functionality; this was due to recombination between highly similar ZFN monomers arranged in tandem.
