Homologous recombination mediated insertion of anti-HBV CRISPR/Cas9 encoding sequences into the helper-dependent adenoviral vector genome

Abstract

Hepatitis B is caused by the hepatitis B virus (HBV), which maintains a stable DNA genome in infected hepatocytes that prolongs infection. Current treatments serve only to reduce the effects of the infection, but do not remove the viral genome from infected cells. As such, gene therapy approaches have been explored to target and eradicate the HBV genome from infected cells. Clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR associated protein 9 (Cas9) has been shown to be effective against HBV, but it is difficult to efficiently deliver these sequences to targeted cells. Viral vectors like adenoviruses are often used as delivery vehicles for gene therapy. Helper-dependent adenoviral vectors (HDAdVs) are widely used, and though they are well suited to target hepatocytes, they have a large genome that is difficult to insert transgenes into. The aim of this study was to design a system to efficiently insert transgenes into the HDAdV genome through homologous recombination with an anti-HBV CRISPR/Cas9-expressing shuttle plasmid. An anti-HBV CRISPR/Cas9 sequence and a nanoLuciferase (nLuc) reporter gene was inserted into a previously constructed shuttle plasmid, that was designed to include two regions that are homologous to regions within the HDAdV genome. Each transgene-expressing shuttle was used with a plasmid expressing the HDAdV genome to co-transform recA expressing Escherichia coli (E. coli) cells, wherein homologous recombination between the two regions of homology was induced. However, restriction analysis of the resultant recombinants indicated significant instability that hindered their characterisation. A low copy ori and a 400 bp fragment of the adenovirus genome associated with increased vector stability was inserted into the shuttle and recombination between this nLuc-expressing shuttle and the HDAdV genome resulted in stable recombinants. These, however, did not include the transgene. This indicated that despite the improved stability of the recombinants, unintended recombination events still occurred that excluded the transgene from the final product. As such, further work must be done to insert the transgene elsewhere in the shuttle to ensure that recombination events include the transgene in the final recombinant. The implications of this work are that one the biggest drawbacks of HDAdV application can be improved to streamline the process and potentially expand on the future use of HDAdVs in gene therapy.