Detection of treatment-resistant infectious HIV after genome-directed antiviral endonuclease therapy

Abstract

Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4+ T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant virus’s RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.

Disruption or Excision of Provirus as an Approach to HIV Cure

“…The induced insertions and deletions introduced into the viral target site may not themselves result in a loss of viral fitness if they maintain the frame of the encoded protein, as illustrated by insertion of a tyrosine after a 3 bp insertion into the viral reverse transcriptase after ZFN therapy. 25 Similar resistance mutants have been shown to emerge and replicate in cultures undergoing treatment with HIV-targeted Cas9. [26][27][28] Fortunately, it appears that the emergence of resistance can be successfully managed in a manner that is analogous to traditional antiviral therapy, by simultaneously targeting multiple sites within the viral genome.…”

Genome editing and the next generation of antiviral therapy

“…De Silva Feelixge et al (2016) were the first to demonstrate that viral resistance can occur following endonuclease therapy. They showed that an insertion mutation introduced into the HIV provirus following ZFN therapy enabled virus replication and ZFN cleavage resistance.…”

“…Although the high reverse transcriptase error rate of HIV makes the likelihood of de novo replication-derived resistance greater than for other DNA viruses, these three studies demonstrate that endonuclease-derived resistance could be a significant concern. Future studies should determine whether endonuclease resistance can occur for other viruses, and if so, whether combinations of endonucleases (De Silva Feelixge et al 2016) or sgRNAs (Kennedy et al 2015) can avoid the development of resistance.…”

Cell and gene therapy strategies to eradicate HIV reservoirs

“…Additional concerns such as the potential immunogenicity of gene editing enzymes, toxicity due to off-target cleavage, and the potential development of treatment resistance will need to be addressed. Indeed, one study identified a mutational insertion in a highly conserved region of pol that provided resistance to subsequent endonuclease cleavage but did not lethally mutate the virus [69]. In another study, virus escaped CRISPR/Cas9 targeting of several conserved regions of the HIV genome [70].…”