Targeted DNA mutagenesis for the cure of chronic viral infections

Abstract

Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches.

Fig 1

Probability of cDNA base pairs within the human genome according to the base pair length of the cleavage enzyme. The calculation assumes random ordering of nucleotides within the human genome.

Fig 2

Structures of cleavage enzymes.

Fig 3

Targeted gene knockout by DNA-editing enzymes. The target sequence (red) is bound by the enzyme, leading to a DNA double-strand break. HE cleavage leaves 3′ overhangs as depicted here; ZFNs and TALENs leave 5′ overhangs. Such DNA double-strand breaks are typically repaired by precise nonhomologous end joining (NHEJ), which restores the original sequence. However, in the continued presence of DNA-editing enzymes, repeated rounds of cleavage and repair ultimately lead to mutagenic NHEJ pathways. In the example shown here, there is a deletion of 16 bp, resulting in a loss of amino acids and a frameshift mutation in the encoded protein. Small deletions such as this are common, although larger deletions and more-complex insertions/deletions can also be observed.