Analysis of Adenovirus-Host Interactions to Improve Recombinant Adenoviral Vectors for Gene Therapy

Recombinant adenoviral vectors are among the most commonly used
vehicles in gene therapy. Replication-deficient adenoviruses
include early generation adenoviruses, which are deleted in less
than three adenoviral genes, and the high-capacity adenoviruses
(HC-AdV) as the most advanced form. HC-AdVs are deleted for all
viral sequences leaving only the two inverted terminal repeat
sequences (ITRs) and the packaging signal from the original viral
genome. Therefore, up to 36 kilobases of foreign DNA can be
packaged by HC-AdV particles and transduced into the desired target
cell. Efficacy of these vectors was shown in several animal models,
in which a single injected virus dose resulted in up to 3 years of
transgene expression. However, also for recombinant adenoviruses
including HC-AdVs limiting factors remain, which were investigated
and improved in the course of this work. The production of HC-AdV
represents one limiting factor because it is a labour intensive and
sophisticated process that requires some experience. Therefore, in
this study, a protocol was developed that simplifies the generation
of these viruses starting with an improved cloning procedure and
ending with precise titration of the purified particles. In
addition, this improved virus production procedure was used to
demonstrate the feasibility of HC-AdV to delivery short-hairpin
RNAs, thus reducing hepatitis B RNA molecules in vitro and in vivo.
The majority of HC-AdVs are currently based on the adenovirus
serotype 5 (Ad5). However, DNA sequences inserted into the HC-AdV
genome and remaining viral sequences were shown to influence
duration and stability of transgene expression, which can
negatively influence the outcome of a therapeutic approach. By
analyzing viral ITR sequences derived from different adenoviral
serotypes, this work demonstrated that ITR-driven transcriptional
activity from several serotypes but also inhibiting functions occur
leading to reduced transgene expression. Furthermore, a negative
impact of ITR sequences on nearby promoters could be observed. The
data obtained in this work suggest that it could be beneficial to
introduce shielding sequences into the HC-AdV genome, which flank
the transgene expression cassettes and therefore, prevent undesired
side effects. Moreover, the results indicated, that pursuing ITRs
from adenovirus serotype 7 in the context of an adenoviral vector
could be advantageous, as it demonstrated most suitable features
regarding transcriptional activation and influence on promoter
performance. The efficiency of HC-AdV in terms of long-term
expression of foreign DNA sequences is mainly based on the
stability of vector genomes in quiescent cells. In dividing cells,
however, a continuous reduction of the viral DNA reduces the
therapeutic effect. Thus, integration systems on the basis of viral
hybrid vectors were developed, which result into
recombinase-mediated somatic integration of the therapeutic DNA
from the HC-AdV genome into the chromosomal DNA. The most prominent
representative of non-viral integration systems is the Sleeping
Beauty (SB) transposase. Although function and efficacy of this
transposase was shown in the context of an HC-AdV, it turned out,
that transgene expression is decreased after Sleeping Beauty
mediated transposition. Herein, analysis of transposition in cells
with suppressed RNA interference pathway, showed a higher
transposition rate in RNA interference knockdown cells compared to
control cells, which was mainly based on an increased transgene
expression. Therefore, this work shows for the first time that due
to convergent transcription, originated from the two SB recognition
sequences (IRs) flanking the transposon, formation of
double-stranded RNAs (dsRNAs) can occur. These dsRNAs can be
substrates for the RNAi mechanism and contribute to the silencing
of gene expression. In the future this finding can be used to
significantly improve the SB transposon technology. Moreover the
influence of the RNAi mechanism on the adenovirus life cycle could
be demonstrated within this project. By the suppression of the RNAi
pathway using an RNAi suppressor protein we could improve
recombinant adenovirus replication and viral particle production,
up to 100-fold. In addition, this RNAi suppressor protein increased
production of HC-AdV up to 6-fold. This upregulation was mainly
based on the increased expression of viral regulatory proteins as
well as the suppression of small adenoviral RNAs. In conclusion,
this work provides different strategies to improve HC-AdVs for gene
therapeutic purposes. Furthermore, it investigated mechanisms that
negatively interfere with the therapeutic outcome, which need to be
considered in future work. In particular, the influence of the RNA
interference pathway on the replication profile of recombinant
adenoviruses could be demonstrated for the first time essentially
broadening the potential of these vectors with respect to viral
production and design of oncolytic adenoviruses. In summary, this
study emphasizes the importance of understanding the biology of
viral vectors systems, which then can be translated into the
development of optimized vectors for gene therapeutic applications.