A High Excision Potential of TALENs for Integrated DNA of HIV-Based Lentiviral Vector

Abstract

DNA-editing technology has made it possible to rewrite genetic information in living cells. Human immunodeficiency virus (HIV) provirus, an integrated form of viral complementary DNA in host chromosomes, could be a potential target for this technology. We recently reported that HIV proviral DNA could be excised from the chromosomal DNA of HIV-based lentiviral DNA-transduced T cells after multiple introductions of a clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 endonuclease system targeting HIV long terminal repeats (LTR). Here, we generated a more efficient strategy that enables the excision of HIV proviral DNA using customized transcription activator-like effector nucleases (TALENs) targeting the same HIV LTR site. A single transfection of TALEN-encoding mRNA, prepared from in vitro transcription, resulted in more than 80% of lentiviral vector DNA being successfully removed from the T cell lines. Furthermore, we developed a lentiviral vector system that takes advantage of the efficient proviral excision with TALENs and permits the simple selection of gene-transduced and excised cells in T cell lines.

The clinical applications of genome editing in HIV

“…82 More conventional anti-HIV nucleases have also been described based on homing endonuclease, ZFNs, TALENs, and CRISPR/Cas9, targeting different regions of the HIV-1 genome, and with a demonstrated ability to reduce HIV-1 content in various cell lines. [83][84][85][86][87][88] Although these studies provide the initial proof of principle that gene disruption could be used to disrupt or excise an integrated HIV-1 genome, significant challenges will exist in the clinical translation of these approaches. No efficient method yet exists to enable in vivo delivery of nucleases to HIV target cells, and delivery to the subset of cells that comprise the latent reservoir will be especially problematic, due both to the location of these cells in inaccessible organ niches and the lack of identifying surface markers of viral infection.…”

Clinical Applications of Genome Editing to HIV Cure

“…Anti-HIV nucleases have been developed based on evolved Cre recombinase, ZFNs, TALENs, and CRISPR/Cas9, targeting different regions of the HIV-1 genome, and have been shown to reduce integrated HIV-1 content in various cell lines. [62][63][64][65][66][67][68][69] Significant challenges exist in the clinical translation of this approach. Targeted nucleases against HIV can promote viral escape and accelerate evolution, observed in culture over an extended period of time.…”

Disruption or Excision of Provirus as an Approach to HIV Cure

“…Importantly, the levels of excision seen have generally exceeded the levels of mutagenesis in single-nuclease approaches. [18][19][20][21] This is presumably because closely spaced pairs of DNA DSBs lead to release of the intervening HIV sequence, making it much less likely that cellular DNA repair mechanisms can restore the intact integrated viral sequence. Although originally observed in the LTR-targeting approaches described earlier, high efficiency of excision can also be achieved by using Cas9 with two or more guide RNAs targeting non-LTR sequences, suggesting that this approach may be widely applicable.…”