Generation of Adenovirus Vectors Devoid of All Viral Genes by Recombination between Inverted Repeats

Abstract

Direct or inverse repeated sequences are important functional features of prokaryotic and eukaryotic genomes. Considering the unique mechanism, involving single-stranded genomic intermediates, by which adenovirus (Ad) replicates its genome, we investigated whether repetitive homologous sequences inserted into E1-deleted adenoviral vectors would affect replication of viral DNA. In these studies we found that inverted repeats (IRs) inserted into the E1 region could mediate predictable genomic rearrangements, resulting in vector genomes devoid of all viral genes. These genomes (termed ΔAd.IR) contained only the transgene cassette flanked on both sides by precisely duplicated IRs, Ad packaging signals, and Ad inverted terminal repeat sequences. Generation of ΔAd.IR genomes could also be achieved by coinfecting two viruses, each providing one inverse homology element. The formation of ΔAd.IR genomes required Ad DNA replication and appeared to involve recombination between the homologous inverted sequences. The formation of ΔAd.IR genomes did not depend on the sequence within or adjacent to the inverted repeat elements. The small ΔAd.IR vector genomes were efficiently packaged into functional Ad particles. All functions for ΔAd.IR replication and packaging were provided by the full-length genome amplified in the same cell. ΔAd.IR vectors were produced at a yield of ∼104 particles per cell, which could be separated from virions with full-length genomes based on their lighter buoyant density. ΔAd.IR vectors infected cultured cells with the same efficiency as first-generation vectors; however, transgene expression was only transient due to the instability of deleted genomes within transduced cells. The finding that IRs present within Ad vector genomes can mediate precise genetic rearrangements has important implications for the development of new vectors for gene therapy approaches.

Adenoviruses as vaccine vectors

“…Vectors deleted of E1, E2A, E4, and E3 [46,47] and vectors lacking all viral genes have been developed for gene therapy. The latter type of constructs, commonly referred to as gutted vectors, can be propagated only in the presence of helper viruses, which commonly contaminate the vector batches [48]. Furthermore, gutted adenoviral constructs can currently not be propagated to quantities needed for clinical development of vectors destined for mass vaccination.…”

“…Alternatively, construction of other serotypes of adenoviral vectors, in which the endogenous E4 ORF6 (which binds to E1B) is replaced by that of AdHu5 virus, may allow for their propagation on available cell lines that provide the E1 of AdHu5 virus in trans [51]. Propagation of adenoviral vectors with additional deletion in either E4 or E2 requires that the deleted genes be provided by the packaging cell lines [46,47] or by a helper virus [48].…”

Biology of Adenovirus and Its Use as a Vector for Gene Therapy

“…To propagate the helper-dependent genome, the presence of a helper virus that provides the functions required for replication and assembly is required, as production of a complementing cell line has not been possible because of the need for high levels of some virion components and the toxicity of some of these proteins to the cell. The main problem to date is the inability to completely separate virions containing the helper-dependent chromosome from those containing the helper virus genome (Steinwaerder et al, 1999;Sandig et al, 2000). Early strategies that were pursued to reduce helper virus contamination included the use of a helper virus carrying a mutated packaging signal, and minimizing the size of the helper-dependent chromosome compared with that of the helper virus with the hope that the two types of virions could be separated on the basis of their different densities.…”

Integrating Adenovirus–Adeno-Associated Virus Hybrid Vectors Devoid of All Viral Genes

“…Previously, we demonstrated that IRs inserted into first-generation Ad vector genomes mediate precise genomic rearrangements resulting in vector genomes devoid of all viral genes that are efficiently packaged into functional Ad capsids (36). We hypothesized that these deleted genomes were formed by intermolecular homologous recombination between the IRs.…”

“…In addition, this hybrid vector should be devoid of all Ad genes whose expression may cause immunological or toxic side effects. On the way to reach this goal, we utilized our earlier finding that inverted repeat sequences (IRs) inserted into Ad vector genomes mediated predictable genomic rearrangements resulting in vector genomes devoid of all viral genes (36). These genomes (named ⌬Ad.IR) contained only the transgene cassette flanked on both sides by precisely duplicated IRs, Ad packaging signals, and Ad ITRs.…”