Excision of HIV 1 DNA by gene editing: a proof of concept in vivo study
A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV 1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV 1 replication in patient derived CD4+ T cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5' LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV 1 genome. Tail vein injection of transgenic mice with a recombinant Adeno associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV 1 DNA and excision of a 978 bp DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, lung and kidney as well as in the circulating lymphocytes. Retro orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof of concept studies, for the first time, demonstrate the in vivo eradication of HIV 1 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA.
Gene Therapy