Adenovirus-based gene therapy approaches for hemophilia B

Gene therapy can be used to treat devastating inherited diseases,
especially diseases and patients that are not suitable for a
conventional cure. The blood clotting disorder hemophilia is one of
the most extensively studied monogenetic diseases in gene
therapeutic approaches. Several viral vectors were tested for the
treatment of hemophilia B. The administration of an episomal
adenoviral vector at non-toxic dose showed effective phenotypic
correction, but the therapeutic effect was only transient.
Therefore, the combination of non-viral integration machineries for
somatic integration with adenoviral vectors for efficient delivery
offers a promising alternative for achieving persistent transgene
expression. Towards this end, the delivery of the Sleeping Beauty
transposase (SB) integration machinery via high-capacity adenoviral
vectors (HC-AdVs) has demonstrated efficient hepatocyte-directed
gene transfer and long-term coagulation factor IX expression in
vivo. However, the safety issues of this adenoviral vector/Sleeping
Beauty transposase (AdV/SB) hybrid-vector system, especially the
vector dose-effect and genotoxicity were not addressed yet. Thus, I
evaluated this hybrid-vector system in both mice and a canine model
for hemophilia B with different vector dose settings, and analyzed
the integration profile in respect to genotoxicity after systemic
administration. First of all, the viral vector preparations
involved in the AdV/SB hybrid-vector studies were analyzed
regarding physical and infectious titers. To avoid toxic side
effects, the helper virus (HV) contamination levels were quantified
and the potential existence of replication-competent adenovirus
(RCA) in final vector preparations was excluded by quantitative
real-time PCR. Only the HC-AdV preparations with high amounts of
transducing units (107-108 TUs/µl), low HV contamination levels
(