A Novel Adenoviral Hybrid-vector System Carrying a Plasmid Replicon for Safe and Efficient Cell and Gene Therapeutic Applications


In dividing cells, the two aims a gene therapeutic approach should accomplish are efficient nuclear delivery and retention of therapeutic DNA. For stable transgene expression, therapeutic DNA can either be maintained by somatic integration or episomal persistence of which the latter approach would diminish the risk of insertional mutagenesis. As most monosystems fail to fulfill both tasks with equal efficiency, hybrid-vector systems represent promising alternatives. Our hybrid-vector system synergizes high-capacity adenoviral vectors (HCAdV) for efficient delivery and the scaffold/matrix attachment region (S/MAR)–based pEPito plasmid replicon for episomal persistence. After proving that this plasmid replicon can be excised from adenovirus in vitro, colony forming assays were performed. We found an increased number of colonies of up to sevenfold in cells that received the functional plasmid replicon proving that the hybrid-vector system is functional. Transgene expression could be maintained for 6 weeks and the extrachromosomal plasmid replicon was rescued. To show efficacy in vivo, the adenoviral hybrid-vector system was injected into C57Bl/6 mice. We found that the plasmid replicon can be released from adenoviral DNA in murine liver resulting in long-term transgene expression. In conclusion, we demonstrate the efficacy of our novel HCAdV-pEPito hybrid-vector system in vitro and in vivo.

There are two major strategies for long-term maintenance of the transgene within a transduced cell of which one is based on somatic integration and the other is based on extrachromosomal maintenance of the therapeutic DNA. Modern adenoviral vectors efficiently deliver the genetic information into the nucleus but due to the absence of efficient nuclear retention and replication, recombinant adenoviral DNA molecules remain episomal and transgene expression levels decline due to the loss of vector molecules in dividing tissue.1,2 So-called hybrid-vector systems are capable of maintaining the administered extrinsic DNA by merging efficient viral delivery with a second component for nuclear persistence of the therapeutic gene. In currently available versions of hybrid vectors, the viral vector encodes an integration machinery of diverse viral or non-viral offspring and the therapeutic DNA to be integrated into the host genome.2,3 Once expressed, the integration apparatus inserts the therapeutic gene into the chromosomes, allowing its duplication during cell cycle and long-term expression. The adenoviral capsid has already been packaged with integration machineries of the AAV rep protein,4,5 the Sleeping Beauty transposase system,6,7 and the bacteriophage-derived integrase PhiC31.8 However, adverse effects such as genotoxicity and insertional mutagenesis may be expected from hybrid vectors using recombinases for somatic integration analogous to integrating retroviral vectors that were demonstrated to result in serious adverse events in clinical trials.9 Furthermore, position effect variegation, for instance integration into transcriptionally inactive regions of the host genome, may result in vanishing expression of the therapeutic transgene. In contrast, hybrid-vector systems that preserve the therapeutic gene in an episomally replicating plasmid within the cell nucleus should not display any risk of insertional mutagenesis.

Toward this end, episomal adenoviral hybrid vectors based on the Epstein–Barr virus (EBV) replication and retention mechanisms have already been described.10,11,12,13 Two of these studies10,11 used high-capacity adenoviral vectors (HCAdV) for delivery of the EBV episome. HCAdV lacks all viral coding sequences and compared with other adenovirus-based vectors HCAdV features the highest packaging capacity for foreign DNA with up to 36 kb.14 Gene therapeutic applications of HCAdV were effectually described for various monogenic diseases.15,16,17,18,19,20 This type of vectors represent one of the most advanced versions for adenoviral gene delivery resulting in reduced toxicity attenuated immune responses in vivo.21,22

Our novel hybrid-vector synergizes the HCAdV technology for efficient delivery and the episomal plasmid replicon pEPito for nuclear retention and replication of the transgene. The pEPito vector23 is a promoter optimized and CpG-depleted derivative of the plasmid pEPI-1, which was constructed in 1999.24,25 Essential feature of both plasmids is a 2 kb S/MAR (scaffold/matrix attachment region) sequence23 that is derived from the 5′-region of the human β-interferon gene26 and comprises 69% of nucleotides A and T, extensive base unpairing regions and >94% binding-affinity to the nuclear matrix. Importantly, upstream transcription directed without any stop-signal into S/MAR is obligatory for the episomal persistence and replication of both pEPI-1 and pEPito, most likely because of conformational changes necessary for the S/MAR to operate.27,28 S/MAR-binding to nuclear matrix proteins and association with early replicating foci that is preserved over mitosis presumably mediates the equal distribution of pEPI-1 to daughter cells during mitosis by non-covalent attachment of the plasmids to metaphase chromosomes.24,29,30 pEPI-1 persists at a copy number of 2–10 per cell over hundreds of generations, and its replication proceeds once per cell cycle in early S phase with the prereplication complex being able to assemble at multiple sites of the plasmid.31 Compared with its ancestor pEPI-1, pEPito shows a stronger and more prolonged transgene expression in vitro and in vivo, although still sharing the characteristics of episomal replication and persistence.23 Attenuated epigenetic silencing and inflammatory response in vivo can be expected due to CpG-depletion of the pEPito promoter and the backbone.