A critical challenge for HIV cure is the presence of latently-infected cells, which are only distinguished from normal uninfected cells by the presence of proviral DNA. Targeted endonucleases (zinc finger nucleases, TAL effector nucleases, homing endonucleases, and CRISPR/Cas proteins) offer the ability to specifically disrupt genomic loci of interest, and in principle such loci could include integrated HIV. Our group and others have now demonstrated that these endonucleases can be used to efficiently disrupt or even excise integrated HIV in vitro, confirming the promise of this approach. Nevertheless, several challenges remain before this approach can disrupt a meaningful proportion of the latent HIV reservoir. We recently demonstrated the in vitro emergence of endonuclease-resistant infectious virus, due to a ZFN-induced insertion in the thumb region of reverse transcriptase, producing a virus that could efficiently replicate and yet was resistant to cleavage by the reverse transcriptase-specific ZFN. This suggests that caution should be exercised in the development of antiviral therapies based on a single nuclease. Furthermore, efficient delivery to cells harboring integrated virus in vivo remains an unsolved problem, which must be addressed before these therapies can move into clinical application. We have packaged antiviral endonucleases into adeno-associated virus vectors, which can efficiently deliver them to T cell lines, and are currently evaluating these in the in vivo setting. Our ongoing work focuses on increasing the efficiency of transgene delivery to relevant subsets of T cells, and maximizing the rate of provirus mutagenesis or excision by targeted endonucleases.
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