No more helper adenovirus: production of gutless adenovirus (GLAd) free of adenovirus and replication-competent adenovirus (RCA) contaminants

Abstract

Gene therapy is emerging as an effective treatment option for various inherited genetic diseases. Gutless adenovirus (GLAd), also known as helper-dependent adenovirus (HDAd), has many notable characteristics as a gene delivery vector for this particular type of gene therapy, including broad tropism, high infectivity, a large transgene cargo capacity, and an absence of integration into the host genome. Additionally, GLAd ensures long-term transgene expression in host organisms owing to its minimal immunogenicity, since it was constructed following the deletion of all the genes from an adenovirus. However, the clinical use of GLAd for the treatment of inherited genetic diseases has been hampered by unavoidable contamination of the highly immunogenic adenovirus used as a helper for GLAd production. Here, we report the production of GLAd in the absence of a helper adenovirus, which was achieved with a helper plasmid instead. Utilizing this helper plasmid, we successfully produced large quantities of recombinant GLAd. Importantly, our helper plasmid-based system exclusively produced recombinant GLAd with no generation of helper plasmid-originating adenovirus and replication-competent adenovirus (RCA). The recombinant GLAd that was produced efficiently delivered transgenes regardless of their size and exhibited therapeutic potential for Huntington’s disease (HD) and Duchenne muscular dystrophy (DMD). Our data indicate that our helper plasmid-based GLAd production system could become a new platform for GLAd-based gene therapy. A new protocol allows for the manufacturing of a next-generation gene therapy vector without contamination of helper adenovirus and replication-competent adenovirus (RCA). Adenoviruses are often used to deliver therapeutic DNA, but their proteins can trigger immune reactions. So-called ‘gutless’ adenoviruses that lack all viral genes don’t cause the same problem but their production has traditionally relied on a helper adenovirus that remains as an unavoidable contaminant. A team led by Dai-Wu Seol from Chung-Ang University in Seoul, South Korea, has now prepared large quantities of gutless adenoviruses using helper plasmid, a circular DNA that encodes all the proteins needed for production of gutless adenoviruses but do not leave behind any contaminant adenoviruses. Gutless adenoviruse vectors made this way successfully delivered corrected copies of the faulty genes responsible for human diseseas into human cells and mice.